HDAC inhibitors and therapeutic methods using the same

ABSTRACT

Histone deacetylases inhibitors (HDACIs) and compositions containing the same are disclosed. Methods of treating diseases and conditions wherein inhibition of HDAC provides a benefit, like a cancer, a neurodegenerative disorder, a peripheral neuropathy, a neurological disease, traumatic brain injury, stroke, hypertension, malaria, an autoimmune disease, autism, autism spectrum disorders, and inflammation, also are disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. provisional patentapplication No. 62/252,064, filed Nov. 6, 2015, and U.S. provisionalpatent application No. 62/213,747, filed Sep. 3, 2015, each incorporatedherein by reference in its entirety.

STATEMENT OF GOVERNMENTAL INTEREST

This invention was made with government support under Grant No.5R43CA133985 awarded by the National Institutes of Health (NIH). Thegovernment has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to histone deacetylase (HDAC) inhibitors,to pharmaceutical compositions comprising one or more of the HDACinhibitors, to methods of increasing the sensitivity of cancer cells tothe cytotoxic effects of radiotherapy and/or chemotherapy comprisingcontacting the cell with one or more of the HDAC inhibitors, and totherapeutic methods of treating conditions and diseases whereininhibition of HDAC provides a benefit, for example, a cancer, aninflammation, a neurological disease, a neurodegenerative disorder,stroke, traumatic brain injury, allograft rejection, autoimmunediseases, and malaria, comprising administering a therapeuticallyeffective amount of a present HDAC inhibitor to an individual in needthereof.

BACKGROUND OF THE INVENTION

Inhibitors of HDACs modulate transcription and induce cell growtharrest, differentiation, and apoptosis. HDAC inhibitors (HDACIs) alsoenhance the cytotoxic effects of therapeutic agents used in cancertreatment, including radiation and chemotherapeutic drugs. Moreover,recent research indicates that transcriptional dysregulation maycontribute to the molecular pathogenesis of certain neurodegenerativedisorders, such as Huntington's disease, Rett syndrome,Charcot-Marie-Tooth disease (CMT) and other peripheral neuropathies,spinal muscular atrophy, amyotropic lateral sclerosis, and ischemia. Forexample, suberoylanilide hydroxamic acid (SAHA) has been shown topenetrate into the brain to dramatically improve motor impairment in amouse model of Huntington's disease, thereby validating researchdirected to HDACIs in the treatment of neurodegenerative diseases.Furthermore, selective HDAC6 inhibitors have been shown to rescue theCMT phenotype, restore proper mitochondrial motility, and correct theaxonal transport defects observed in transgenic mice. Selective HDAC6inhibitors also induce the re-innervation of muscles and increase thenumber of observed neuromuscular junctions in these same models (C.d'Ydewalle et al., Nature Medicine 2011).

A recent review summarized evidence that aberrant histoneacetyltransferase (HAT) and HDAC activity may be a common underlyingmechanism contributing to neurodegeneration. Moreover, from a mousemodel of depression, the therapeutic potential of HDACs in treatingdepression is discussed. See WO 2008/019025, designating the UnitedStates, incorporated herein in its entirety.

Eleven isozymes in the HDAC family of enzymes, which can be grouped intoclasses by their evolutionary relationships, have been identified.Structure and function appear to be conserved among members of thevarious classes. The HDAC family is made up of class I HDACs, includingHDAC1, 2, 3, and 8; class IIa, including HDAC4, 5, 7, and 9; class IIb,including HDAC6 and 10; and a class IV enzyme, HDAC11 (A. J. de Ruijteret al., The Biochemical Journal 2003, 370(Pt), 737-749).

The class I HDACs are found primarily in the nucleus and are expressedin all tissue types, except for the muscle cell-specific HDAC8. Theclass I HDACs interact with many key transcription factors regulatinggene expression, including CoREST and NuRD. Class IIa HDACs have tissuespecific expression, and are found in both the nucleus and cytoplasm.Unlike the other isozymes, the class IIb HDAC6 does not extensivelyassociate with transcription factors, and acts as a deacetylase onnon-histone proteins, including α-tubulin, HSP90, cortactin, and theperoxiredoxins (O. Witt et al., Cancer Letters 2008; R. B. Parmigiana etal., PNAS 2008).

HDACs form multiprotein complexes with many regulatory proteins insidethe cell. For example, HDAC4, 5, and 7 actually lack intrinsicdeacetylase ability, and gain activity only by interacting with HDAC3.Each isozyme interacts with a specific series of regulatory proteins andtranscription factors and has a specific set of substrates, and thuseach regulates a specific series of genes and proteins (O. Witt et al.,Cancer Letters 2008). The design of selective HDAC isozyme inhibitorsallows preferential inhibition of only the isozyme(s) relevant to aparticular disease or condition, thereby reducing the probability ofcounterproductive and/or adverse effects resulting from an unwanted andundesired inhibition of other HDAC isozymes.

HDAC6 is the most abundant histone deacetylase isozyme in the humanbody, and along with HDAC7, is the most commonly expressed isozyme inthe brain (A. J. de Ruijter et al., The Biochemical Journal 2003,370(Pt), 737-749). Structurally significant features of HDAC6 includetwo deacetylase domains and a zinc finger motif. It is most commonlyfound in the cytoplasm, but can be shuttled into the nucleus via itsnuclear export signal. A cytoplasmic retention signal, which sequestersthe enzyme in the cytoplasm, also was found (A. Valenzuela-Fernandez etal., Trends in Cell Biology 2008, 18(6), 291-297). The functions ofHDAC6 are unlike any of the other HDAC isozymes. Many non-histonesubstrates are deacetylated by HDAC6, including α-tubulin, HSP90,cortactin, and peroxiredoxins (O. Witt et al., Cancer Letters 2008; R.B. Parmigiani et al., PNAS USA 2008, 105(28), 9633-9638).

The design of HDACIs focuses on the three major domains of the enzymemolecule. A zinc binding group (ZBG) of the HDACI typically is ahydroxamic acid, benzamide, or thiol, although other functional groupshave been used. This ZBG moiety of the inhibitor chelates the zinccofactor found in the active site of the enzyme. The ZBG moietytypically is bonded to a lipophilic linker group, which occupies anarrow channel leading from the HDAC surface to the active site. Thislinker, in turn, is bonded to a surface recognition, or ‘cap’, moiety,which typically is an aromatic group that interacts with residues at thesurface of the enzyme (K. V. Butler et al., Current PharmaceuticalDesign 2008, 14(6), 505-528).

Consideration of each structural element is important in the design ofHDACIs (37). Alteration of the ZBG has profound effects on inhibitorpotency. The most potent inhibitors are hydroxamates, though compoundsbearing different ZBG groups such as ketones, amides, and thiols canalso effectively inhibit the enzyme. Low molecular weight compoundshaving carboxylic acid ZBGs, such as valproic acid (VPA), inhibit HDACsat micromolar potency, but have profound effects in vivo when given inhigh doses. Hydroxamic acids chelate zinc in a bidentate fashion andhydrogen bond with H142 and H143 (HDAC8), as revealed by crystalstructure data. Computational studies have refined this description ofchelation, demonstrating that the hydroxamic acid carbonyl coordinateszinc more strongly than the hydroxyl group.

The linker region typically is a hydrophobic aryl or alkyl scaffold,which occupies the hydrophobic HDAC catalytic channel. Most reportedHDACIs, including SAHA, feature an alkyl chain linker, mimicking thelysine alkyl chain. Aromatic groups are frequently included in thelinker region of an HDACI, for example, in the HDACIs panbinostat andbelinostat.

Manipulation of the cap group can greatly increase potency because thisgroup has the potential to interact with multiple residues at the enzymesurface. The most potent inhibitors typically feature an aryl cap groupscaffold. Biaryl and heteroaromatic cap group scaffolds have beenextensively investigated. The large tetrapeptide motif of apicidinimparts high potency, and gives high potency with a number of diverseZBGs. The tetrapeptide cap group motif is common to natural productHDACIs, and has been engineered to produce libraries of tetrapeptideHDACIs for use in screening protocols.

Currently, at least eleven HDACIs are in clinical development. TheseHDACIs can be divided into at least five chemical classes, illustratedbelow, based on their structure, and in most cases they broadly andnonselectively inhibit class I/II HDACs with varying efficiency. Thesefive chemical classes are hydroxamates, cyclic tetrapeptides, cyclicpeptides, short-chain fatty acids, and benzamides. Typically, knownHDACIs fail to show prominent HDAC isozyme selectivity, which as statedabove can cause serious problems in a clinical setting, especially inthe treatment of diseases and conditions wherein a prolonged drugadministration of an HDACI is required. For example, it has been foundthat some HDACIs enhance lung and microglial inflammation (TSA andSAHA), as well as high glucose-induced inflammation. If this effect islinked to specific HDAC isozymes, the use of certain HDACIs would becontraindicated in various diseases and conditions, such as diabetes andasthma.

The following table summarizes some HDACIs that presently are inclinical trials.

TABLE I Inhibitor Indications SAHA T-cell lymphoma (Approved) RomidepsinT-cell lymphoma (Approved) Multiple myeloma (Phase III) PeripheralT-cell lymphoma (Phase III) Refractory renal cell cancer (Phase II)Valproic Bipolar disorder (Approved) Acid (VPA) Acute myeloid leukemia(Phase I/II with all trans- retinoic acid) PCI-24781 Leukemia (PhaseI/II) ITF-2357 Hodgkins lymphoma (Phase II) Follicular lymphoma (PhaseIII, with yttrium-90- ibritumomab) Juvenile arthritis (Phase II)Myeloproliferative Diseases (Phase II) MS-275 Melanoma Lymphoma (halteddue to dose limiting toxicities) Advanced acute leukemias (Phase 1)Combination trials with DNA methyltransferase inhibitors and5-azacitidine in non-small cell lung cancer (Preclinical) PanbinostatT-cell lymphoma (Phase II) Prostate cancer (Phase I with docetaxel)Belinostat Solid tumors (Phase I) Mesothelioma (Abandoned) MGCD0103Solid tumors (Phase II with gemcitabine) Diffuse large B-cell lymphoma(Phase II) EVP-0334 Parkinson's disease (Phase I)

Clinical trial information relating to HDACIs is published in J. Tan etal., Journal of hematology & oncology. 3:5 (2010) and L. Wang et al.,Nat Rev Drug Discov. 8:969-81 (2009).

HDAC-regulated factors have been implicated in the mechanisms of majorcentral nervous system (CNS) disorders. In Parkinson's disease (PD),α-synuclein binds to histones and inhibits HAT activity, causingneurodegeneration. Application of HDACIs to PD neurons blocksα-synuclein toxicity. Dysregulation of histone acetylation, involvingCBP, a neuroprotective transcription factor with histoneacetyltransferase activity, has been found in Huntington's disease (HD),Alzheimer's disease (AD), and Rubinstein-Taybi syndrome (T. Abel et al.,Curr. Opin. in Pharmacol. 2008, 8(1), 57-64). In a cellular model of AD,cell death was accompanied by loss of CBP function and histonedeacetylation. The mutant HD protein, htt, interacts with CBP,inhibiting the HAT activity and causing cell death. Treatment with anHDACI helps to restore histone acetylation, protecting againstneurodegeneration and improving motor performance in a mouse model of HD(C. Rouaux et al., Biochem. Pharmacol. 2004, 68(6), 1157-1164).

Various studies directed to the application of HDACIs in the context ofCNS disorders have implicated the class II HDACs, particularly HDAC6, aspotential therapeutic targets. One investigation revealed thatinhibition of HDAC6 could be beneficial as a treatment for HD, a diseasefor which no pharmacological treatment is available. The mutant httprotein found in HD disrupts intracellular transport of the pro-survivaland pro-growth nerve factor, BDNF, along the microtubule network,causing neuronal toxicity. Inhibition of HDAC6 promotes transport ofBDNF by promoting tubulin hyperacetylation. TSA (trichostatin A), anonselective HDAC inhibitor, was found to facilitate transport andrelease of BNDF-containing vesicles (J. P. Dompierre et al., J Neurosci2007, 27(13), 3571-3583). These results provide a biological basis forthe identification and development of HDACIs, and particularly HDAC6selective inhibitors, as a treatment for HD and other neurodegenerativedisorders.

HDACIs prevent or delay neuronal dysfunction and death in in vitro andin vivo models thereby indicating that HDACIs are broadlyneuroprotective. For example, HDACIs have shown therapeutic efficacy inthe polyglutamine-expansion disorder Huntington's disease. While theneuroprotective mechanisms of the HDACIs in rodent models are not yetunderstood, it is clear that HDACIs induce the expression of certaingenes that confer neuroprotection. The upregulation of HSP-70 and Bcl-2through the inhibition of HDAC has been observed in the cortex andstriatum of rats after focal cerebral ischemia. HSP-70 expression hasbeen found to result in neuroprotection in a number of disease modelsincluding Alzheimer's disease (AD), Parkinson's disease (PD), andHuntington's disease (HD). In addition, HDAC6 inhibition leads to theacetylation of peroxiredoxin and increases its antioxidant activitywhich may contribute to the neuroprotective effects of these compounds(R. B. Parmigiana et al., PNAS 2008).

Studies also provide good evidence that HDACI-induced p21cip1/waf1expression may play a significant role in HDACI-mediatedneuroprotection. It recently was reported that p21cip1/waf1overexpression protects neurons from oxidative stress-induced death,that p21cip1/waf1 is induced in the rodent brain by HDAC inhibition, andthat homozygous loss of p21cip1/waf1 exacerbates damage in a mouseMCAO/reperfusion model of ischemic stroke. In a similar study, the HDACinhibitor TSA was shown to increase gelsolin expression in neurons, andthat gelsolin expression is necessary for neuroprotection in anoxygen/glucose deprivation model of neurodegeneration and a mouseMCAO/reperfusion model of ischemic stroke.

Alternatively, unrelated to histone acetylation and gene upregulation,proteins such as α-tubulin and HSP90 are targets for acetylation andbecome acetylated when HDACs are inhibited. In tumor cells, theacetylation of HSP90 has been shown to decrease the ability of HSP90 tointeract with certain client proteins and thereby abrogate chaperonefunction. With regard to stroke and traumatic brain injury (TBI), aswell as several other neurodegenerative diseases, the inhibition ofHSP90 is predicted to have a positive effect on neuronal survival.Indeed, the pharmacological HSP90 inhibitor, Geldanamycin, and itsanalogs have been shown to be neuroprotective in a number of strokemodels. HSP90 inhibition and the consequent release of heat-shock factor(HSF) to the nucleus may also, in part, explain an upregulation of HSP70in the brain during focal ischemia and HDACI treatment.

In addition, HDACIs are useful in the treatment of cancers. For example,histone acetylation and deacetylation play important roles in chromatinfolding and maintenance (Kornberg et al., Bjorklund et al., Cell, 1999,96:759-767; Struhl et al., Cell, 1998, 94:1-4). Acetylated chromatin ismore open and has been implicated in the increased radiationsensitivities observed in some cell types (Oleinick et al., Int. J.Radiat. Biol. 1994, 66:523-529). Furthermore, certainradiation-resistant human cancer cells treated with the HDACI inhibitorTSA were sensitized to the damaging effects of ionizing radiation. Thus,HDACIs appear useful as radiation sensitizing agents.

WO 2008/055068 discloses numerous diseases and conditions treatable byHDACIs, including the underlying science and reasoning supporting suchtreatments.

HDAC6 therefore has emerged as an attractive target for drug developmentand research. (C. M. Grozinger et al., Proc. Natl. Acad. Sci. USA 1999,96, 4868-73; and C. Boyault et al., Oncogene 2007, 26, 5468-76.)Presently, HDAC6 inhibition is believed to offer potential therapies forautoimmunity, cancer, and many neurodegenerative conditions. (S. Minucciet al., Nat. Rev. Cancer. 2006, 6, 38-51; L. Wang et al., Nat. Rev. DrugDiscov. 2009, 8, 969-81; J. P. Dompierre et al., J. Neurosci. 2007, 27,3571-83; and A. G. Kazantsev et al., Nat. Rev. Drug Discov. 2008, 7,854-68.) Selective inhibition of HDAC6 by small molecule or genetictools has been demonstrated to promote survival and re-growth of neuronsfollowing injury, offering the possibility for pharmacologicalintervention in both CNS injury and neurodegenerative conditions. (M. A.Rivieccio et al., Proc. Natl. Acad. Sci. USA 2009, 106, 19599-604.)Unlike other histone deacetylases, inhibition of HDAC6 does not appearto be associated with any toxicity, making it an excellent drug target.(O. Witt et al., Cancer Lett 2009, 277, 8-21.) Tubacin, an HDAC6selective inhibitor, used in models of disease, has helped to validate,in part, HDAC6 as a drug target, but its non-drug-like structure, highlipophilicity (ClogP=6.36 (KOWWIN)) and tedious synthesis make it moreuseful as a research tool than a drug. (S. Haggarty et al., Proc. Natl.Acad. Sci. USA 2003, 100, 4389-94.) Other compounds also have a modestpreference for inhibiting HDAC6. (S. Schafer et al., ChemMedChem 2009,4, 283-90; Y. Itoh et al., J. Med. Chem. 2007, 50, 5425-38; and S. Mankuet al., Bioorg. Med. Chem. Lett. 2009, 19, 1866-70.)

In summary, extensive evidence supports a therapeutic role for HDACIs inthe treatment of a variety of conditions and diseases, such as cancersand CNS diseases and degenerations. However, despite exhibiting overallbeneficial effects, like beneficial neuroprotective effects, forexample, HDACIs known to date have little specificity with regard toHDAC inhibition, and therefore inhibit all zinc-dependent histonedeacetylases. It is still unknown which is (are) the salient HDAC(s)that mediate(s) neuroprotection when inhibited. Emerging evidencesuggests that at least some of the HDAC isozymes are absolutely requiredfor the maintenance and survival of neurons, e.g., HDAC1. Additionally,adverse side effect issues have been noted with nonspecific HDACinhibition. Thus, the clinical efficacy of present-day nonspecificHDACIs for stroke, neurodegenerative disorders, neurological diseases,and other diseases and conditions ultimately may be limited. It isimportant therefore to design, synthesize, and test compounds capable ofserving as potent, and preferably isozyme-selective, HDACIs that areable to ameliorate the effects of neurological disease,neurodegenerative disorder, traumatic brain injury, cancer,inflammation, malaria, autoimmune diseases, immunosuppressive therapy,and other conditions and diseases mediated by HDACs.

An important advance in the art would be the discovery of HDACIs, andparticularly selective HDAC6 inhibitors, that are useful in thetreatment of diseases wherein HDAC inhibition provides a benefit, suchas cancers, neurological diseases, traumatic brain injury,neurodegenerative disorders and other peripheral neuropathies, stroke,hypertension, malaria, allograft rejection, rheumatoid arthritis, andinflammations. Accordingly, a significant need exists in the art forefficacious compounds, compositions, and methods useful in the treatmentof such diseases, alone or in conjunction with other therapies used totreat these diseases and conditions. The present invention is directedto meeting this need.

SUMMARY OF THE INVENTION

The present invention relates to HDACIs, pharmaceutical compositionscomprising the HDACIs, and methods of treating diseases and conditionswherein inhibition of HDAC provides a benefit, such as a cancer, aneurological disease, a neurodegenerative disorder, a peripheralneuropathy, stroke, hypertension, an inflammation, traumatic braininjury, rheumatoid arthritis, allograft rejection, autoimmune diseases,and malaria, comprising administering a therapeutically effective amountof an HDACI to an individual in need thereof. The present invention alsorelates to a method of increasing the sensitivity of a cancer cell toradiotherapy and/or chemotherapy. The present invention also allows forthe use of these HDACIs inhibitors in combination with other drugsand/or therapeutic approaches. In some embodiments, the present HDACIsexhibit selectivity for particular HDAC isozymes, such as HDAC6, overother HDAC isozymes.

More particularly, the present invention relates to HDACIs having astructural formula (I):

wherein A is selected from the group consistingof —C(═O)NHheteroaryl, —NHC(═O)heteroaryl, aryl, heteroaryl, and

wherein B and C, independently, are aryl or heterocyclyl;n is 0-4;p is 0-3; andm is 0 or 1,or a pharmaceutically acceptable salt, hydrate, prodrug thereof.

In another embodiment, the present invention provides a method oftreating a condition or disease by administering a therapeuticallyeffective amount of a present HDACI to an individual in need thereof.The disease or condition of interest is treatable by inhibition of HDAC,for example, a cancer, a neurodegenerative disorder, a traumatic braininjury, a neurological disease, peripheral neuropathy, an inflammation,stroke, hypertension, an autoimmune disease, allograft rejection, andmalaria.

Another embodiment of the present invention provides a method oftreating a cancer comprising administering to an individual in needthereof, such as a human, a therapeutically effective amount of apresent HDACI. A present HDACI can be administered as the soleanticancer therapy or in conjunction with a therapeutically effectiveamount of a second anticancer agent, such as a PARP inhibitor, radiationand/or chemotherapy.

Another embodiment of the present invention provides a method ofincreasing the sensitivity of a cancer cell to the cytotoxic effects ofradiotherapy and/or chemotherapy comprising contacting the cell with aneffective amount of a present HDACI. In certain embodiments, the cell isan in vivo cell.

In another embodiment, the present invention provides a method oftreating a neurological disease comprising administering to anindividual in need thereof, such as a human, a therapeutically effectiveamount of a present HDACI. The present invention also relates to amethod of treating neurodegenerative disorders, peripheral neuropathies,and traumatic brain injuries comprising administering a therapeuticallyeffective amount of a present HDACI to an individual in need thereof. Ineach embodiment, a present HDACI can be the sole therapeutic agent orcan be administered with additional therapeutic agents known to treatthe disease or condition of interest.

The present invention also provides a method of treating malaria andother parasitic infections comprising administering a therapeuticallyeffective amount of a present HDACI to an individual in need thereof. Incertain embodiments, the individual is a human. In certain embodiments,said method further comprises optionally coadministering a secondantimalarial compound (e.g., chloroquine).

In yet another embodiment, the present invention provides a method ofinducing immunosuppression in an individual comprising administration ofa therapeutically effective amount of a present HDACI to an individualin need thereof, for example, an individual receiving a transplant. Thismethod further comprises optionally coadministering a secondimmunosuppressant (e.g., cyclosporin) or therapeutic agent.

In still another embodiment, the present invention provides a method oftreating inflammatory diseases and conditions, e.g., arthritis andrheumatic diseases, comprising administration of a therapeuticallyeffective amount of a present HDACI to an individual in need thereof.The method further contemplates optional coadministration of a secondanti-inflammatory drug or therapeutic agent.

In another embodiment, the present invention also provides apharmaceutical composition comprising a present HDACI and apharmaceutically acceptable excipient.

Another embodiment of the present invention is to utilize a presentHDACI and an optional second therapeutically active agent in a method oftreating an individual for a disease or condition wherein inhibition ofHDAC provides a benefit.

In a further embodiment, the invention provides for use of a compositioncomprising a present HDACI and an optional second therapeutic agent forthe manufacture of a medicament for treating a disease or condition ofinterest, e.g., a cancer, neurodegeneration, or autoimmunity.

Still another embodiment of the present invention is to provide a kitfor human pharmaceutical use comprising (a) a container, (b1) a packagedcomposition comprising a present HDACI, and, optionally, (b2) a packagedcomposition comprising a second therapeutic agent useful in thetreatment of a disease or condition of interest, and (c) a packageinsert containing directions for use of the composition or compositions,administered simultaneously or sequentially, in the treatment of thedisease or condition of interest.

A present HDACI and the second therapeutic agent can be administeredtogether as a single-unit dose or separately as multi-unit doses,wherein a present HDACI is administered before the second therapeuticagent, or vice versa. It is envisioned that one or more dose of apresent HDACI and/or one or more dose of a second therapeutic agent canbe administered.

In one embodiment, a present HDACI and a second therapeutic agent areadministered simultaneously. In related embodiments, a present HDACI andsecond therapeutic agent are administered from a single composition orfrom separate compositions. In a further embodiment, a present HDACI anda second therapeutic agent are administered sequentially. A presentHDACI can be administered in an amount of about 0.005 to about 500milligrams per dose, about 0.05 to about 250 milligrams per dose, orabout 0.5 to about 100 milligrams per dose.

Compounds of the invention inhibit HDAC and are useful research toolsfor in vitro study of histone deacetylases and their role in biologicalprocesses.

These and other novel aspects of the present invention will becomeapparent from the following detailed description of the preferredembodiments.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A illustrates the accumulation of acetylated alpha-tubulin aftertreatment of pancreatic cancer cells by present compounds 10, 9, 11, 12,14, 18, and 15;

FIG. 1B illustrates the cell cycle arrest induced by treatment PANC1cells with present compounds 14, 18, and 15;

FIG. 2 contains photographs of PANC1 cells treated with presentcompounds 9 and 6 taken 24 and 48 hours (post wounding) after a scratchwas made through a confluent layer of cells; and

FIG. 3 contains photographs and Western immunoblots showing that HDACinhibitors of present invention induce apoptosis on L3.6p1 pancreaticcancer cells. L3.6p1 pancreatic cancer cells were treated with 10 μM ofcompound 1, 2 or 4 for 24 hrs as indicated. Cells were collected andprocessed to Hoechst staining (upper panel) and cell lysates wereprepared for Western immunoblotting (lower panel). 50 μg of proteinswere separated by SDS-PAGE, transferred to PVDF membrane, andimmunoblotted as indicated.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is directed to novel HDACIs and their use intherapeutic treatments of, for example, cancers, inflammations,traumatic brain injuries, neurodegenerative disorders, neurologicaldiseases, peripheral neuropathies, strokes, hypertension, autoimmunediseases, inflammatory diseases, and malaria. The present HDACIs alsoincrease the sensitivity of a cancer cell to the cytotoxic effects ofradiotherapy and/or chemotherapy. In some embodiments, the presentHDACIs selectively inhibit HDAC6 over other HDAC isozymes.

The present invention is described in connection with preferredembodiments. However, it should be appreciated that the invention is notlimited to the disclosed embodiments. It is understood that, given thedescription of the embodiments of the invention herein, variousmodifications can be made by a person skilled in the art. Suchmodifications are encompassed by the claims below.

The term “a disease or condition wherein inhibition of HDAC provides abenefit” pertains to a condition in which HDAC and/or the action of HDACis important or necessary, e.g., for the onset, progress, expression ofthat disease or condition, or a disease or a condition which is known tobe treated by an HDAC inhibitor (such as, e.g., TSA,pivalolyloxymethylbutane (AN-9; Pivanex), FK-228 (Depsipeptide),PXD-101, NVP-LAQ824, SAHA, MS-275, and or MGCD0103). Examples of suchconditions include, but are not limited to, cancer, psoriasis,fibroproliferative disorders (e.g., liver fibrosis), smooth muscleproliferative disorders (e.g., atherosclerosis, restenosis),neurodegenerative diseases (e.g., Alzheimer's, Parkinson's, Huntington'schorea, amyotropic lateral sclerosis, spino-cerebellar degeneration,Rett syndrome), peripheral neuropathies (Charcot-Marie-Tooth disease,Giant Axonal Neuropathy (GAN)), inflammatory diseases (e.g.,osteoarthritis, rheumatoid arthritis, colitis), diseases involvingangiogenesis (e.g., cancer, rheumatoid arthritis, psoriasis, diabeticretinopathy), hematopoietic disorders (e.g., anemia, sickle cell anemia,thalasseimia), fungal infections, parasitic infections (e.g., malaria,trypanosomiasis, helminthiasis, protozoal infections), bacterialinfections, viral infections, and conditions treatable by immunemodulation (e.g., multiple sclerosis, autoimmune diabetes, lupus, atopicdermatitis, allergies, asthma, allergic rhinitis, inflammatory boweldisease; and for improving grafting of transplants). One of ordinaryskill in the art is readily able to determine whether a compound treatsa disease or condition mediated by HDAC for any particular cell type,for example, by assays which conveniently can be used to assess theactivity of particular compounds.

The term “second therapeutic agent” refers to a therapeutic agentdifferent from a present HDACI and that is known to treat the disease orcondition of interest. For example, when a cancer is the disease orcondition of interest, the second therapeutic agent can be a knownchemotherapeutic drug, like taxol, PARP inhibitors, or radiation, forexample.

The term “HDAC” refers to a family of enzymes that remove acetyl groupsfrom a protein, for example, the ε-amino groups of lysine residues atthe N-terminus of a histone. The HDAC can be a human HDAC, including,HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10,and HDAC11. The HDAC also can be derived from a protozoal or fungalsource.

HDAC inhibitors (HDACIs) typically contain three structural elementswhich are analogous to the structure of acetyllysine. These threestructural elements are a zinc binding group (M), which is responsiblefor chelation of zinc in the active site, a linker region (L), whichbinds to the hydrophobic channel that connects the active site to theouter enzyme surface, and a capping group (Cap), which interacts withresidues at the outer enzyme surface.

As used herein, the terms “treat,” “treating,” “treatment,” and the likerefer to eliminating, reducing, relieving, reversing, and/orameliorating a disease or condition and/or symptoms associatedtherewith. Although not precluded, treating a disease or condition doesnot require that the disease, condition, or symptoms associatedtherewith be completely eliminated, including the treatment of acute orchronic signs, symptoms and/or malfunctions. As used herein, the terms“treat,” “treating,” “treatment,” and the like may include “prophylactictreatment,” which refers to reducing the probability of redeveloping adisease or condition, or of a recurrence of a previously-controlleddisease or condition, in a subject who does not have, but is at risk ofor is susceptible to, redeveloping a disease or condition or arecurrence of the disease or condition, “treatment” therefore alsoincludes relapse prophylaxis or phase prophylaxis. The term “treat” andsynonyms contemplate administering a therapeutically effective amount ofa compound of the invention to an individual in need of such treatment.A treatment can be orientated symptomatically, for example, to suppresssymptoms. It can be effected over a short period, be oriented over amedium term, or can be a long-term treatment, for example within thecontext of a maintenance therapy.

The term “therapeutically effective amount” or “effective dose” as usedherein refers to an amount of the active ingredient(s) that, whenadministered, is (are) sufficient, to efficaciously deliver the activeingredient(s) for the treatment of condition or disease of interest toan individual in need thereof. In the case of a cancer or otherproliferation disorder, the therapeutically effective amount of theagent may reduce (i.e., retard to some extent and preferably stop)unwanted cellular proliferation; reduce the number of cancer cells;reduce the tumor size; inhibit (i.e., retard to some extent andpreferably stop) cancer cell infiltration into peripheral organs;inhibit (i.e., retard to some extent and preferably stop) tumormetastasis; inhibit, to some extent, tumor growth; reduce HDAC signalingin the target cells; and/or relieve, to some extent, one or more of thesymptoms associated with the cancer. To extent the administered compoundor composition prevents growth and/or kills existing cancer cells, itmay be cytostatic and/or cytotoxic.

The term “container” means any receptacle and closure therefor suitablefor storing, shipping, dispensing, and/or handling a pharmaceuticalproduct.

The term “insert” means information accompanying a pharmaceuticalproduct that provides a description of how to administer the product,along with the safety and efficacy data required to allow the physician,pharmacist, and patient to make an informed decision regarding use ofthe product. The package insert generally is regarded as the “label” fora pharmaceutical product.

“Concurrent administration,” “administered in combination,”“simultaneous administration,” and similar phrases mean that two or moreagents are administered concurrently to the subject being treated. By“concurrently,” it is meant that each agent is administered eithersimultaneously or sequentially in any order at different points in time.However, if not administered simultaneously, it is meant that they areadministered to an individual in a sequence and sufficiently close intime so as to provide the desired therapeutic effect and can act inconcert. For example, a present HDACI can be administered at the sametime or sequentially in any order at different points in time as asecond therapeutic agent. A present HDACI and the second therapeuticagent can be administered separately, in any appropriate form and by anysuitable route. When a present HDACI and the second therapeutic agentare not administered concurrently, it is understood that they can beadministered in any order to a subject in need thereof. For example, apresent HDACI can be administered prior to (e.g., 5 minutes, 15 minutes,30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks,5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, orsubsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours,96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks,or 12 weeks after) the administration of a second therapeutic agenttreatment modality (e.g., radiotherapy), to an individual in needthereof. In various embodiments, a present HDACI and the secondtherapeutic agent are administered 1 minute apart, 10 minutes apart, 30minutes apart, less than 1 hour apart, 1 hour apart, 1 hour to 2 hoursapart, 2 hours to 3 hours apart, 3 hours to 4 hours apart, 4 hours to 5hours apart, 5 hours to 6 hours apart, 6 hours to 7 hours apart, 7 hoursto 8 hours apart, 8 hours to 9 hours apart, 9 hours to 10 hours apart,10 hours to 11 hours apart, 11 hours to 12 hours apart, no more than 24hours apart or no more than 48 hours apart. In one embodiment, thecomponents of the combination therapies are administered at 1 minute to24 hours apart.

The use of the terms “a”, “an”, “the”, and similar referents in thecontext of describing the invention (especially in the context of theclaims) are to be construed to cover both the singular and the plural,unless otherwise indicated. Recitation of ranges of values herein merelyserve as a shorthand method of referring individually to each separatevalue falling within the range, unless otherwise indicated herein, andeach separate value and subrange is incorporated into the specificationas if it were individually recited herein. The use of any and allexamples, or exemplary language (e.g., “such as” and “like”) providedherein, is intended to better illustrate the invention and is not alimitation on the scope of the invention unless otherwise claimed. Nolanguage in the specification should be construed as indicating anynon-claimed element as essential to the practice of the invention.

In particular, the present invention is directed to HDACIs, compositionscomprising a present HDACI, and therapeutic uses of the HDACIs of thefollowing structural formula (I):

-   -   wherein A is selected from the group consisting of        —C(═O)NHheteroaryl,    -   —NHC(═O)heteroaryl, aryl, heteroaryl, and

-   -   wherein B and C, independently, are aryl or    -   heterocyclyl and n is 0-4;    -   or a pharmaceutically acceptable salt, hydrate, prodrug thereof.

wherein A is selected from the group consistingof, —NHC(═O)alkyl, —NHC(═O)cycloalkyl, —NHC(═O)heteroaryl, aryl,heteroaryl andn is 0 or 1;p is 0 or 1,or a pharmaceutically acceptable salt, hydrate, prodrug thereof.

Compounds of the present invention inhibit HDAC and are useful in thetreatment of a variety of diseases and conditions. In particular, thepresent HDACIs are used in methods of treating a disease or conditionwherein inhibition of HDAC provides a benefit, for example, cancers,neurological diseases, neurodegenerative conditions, peripheralneuropathies, autoimmune diseases, inflammatory diseases and conditions,stroke, hypertension, traumatic brain injury, autism, and malaria. Themethods comprise administering a therapeutically effective amount of apresent HDACI to an individual in need thereof.

The present methods also encompass administering a second therapeuticagent to the individual in addition to a present HDACI. The secondtherapeutic agent is selected from agents, such as drugs and adjuvants,known as useful in treating the disease or condition afflicting theindividual, e.g., a chemotherapeutic agent and/or radiation known asuseful in treating a particular cancer.

As used herein, the term “alkyl” refers to straight chained and branchedsaturated hydrocarbon groups, nonlimiting examples of which includemethyl, ethyl, and straight chain and branched propyl, butyl, pentyl,hexyl, heptyl, and octyl groups containing the indicated number ofcarbon atoms. The term C_(n) means the alkyl group has “n” carbon atoms.

The term “alkylene” refers to a bidentate moiety obtained by removingtwo hydrogen atoms from an alkane. An “alkylene” is positioned betweentwo other chemical groups and serves to connect them. An example of analkylene group is —(CH₂)_(n)—. An alkyl, e.g., methyl, or alkylene,e.g., —CH₂CH₂—, group can be substituted, independently, with one ormore of halo, trifluoromethyl, trifluoromethoxy, hydroxy, alkoxy, nitro,cyano, alkylamino, and amino groups, for example.

The term “alkenyl” is defined identically as “alkyl,” except forcontaining a carbon-carbon double bond, e.g., ethenyl, propenyl, andbutenyl. The term “alkenylene” is defined identically to “alkylene”except for containing a carbon-carbon double bond. The term“alkdienylene” is defined identically as “alkenylene” except the groupcontains two carbon-carbon double bonds, either conjugated ornon-conjugated.

The term “heteroalkyl” refers to an alkyl group having one or more, andtypically one to three, heteroatoms in the carbon chain of the alkylgroup. The heteroatoms, independently, are selected from O, S, and NR,wherein R is hydrogen, alkyl, cycloalkyl, heterocycloalkyl, aryl, andheteroaryl. A term such as “C₁₋₆heteroalkyl” means that the groupcontains 1 to 6 carbon atoms in addition to the heteroatoms.

The term “perfluoroalkyl” is defined as an alkyl group wherein allhydrogen atoms are replaced by fluorine atoms.

As used herein, the term “halo” and “Hal” are defined as fluoro, chloro,bromo, and iodo.

The term “hydroxy” is defined as —OH.

The term “alkoxy” is defined as —OR, wherein R is alkyl. The term“perfluoroalkoxy” is defined as an alkoxy group wherein all hydrogenatoms are replaced by fluorine atoms.

The term “amino” is defined as —NR₂, wherein each R group,independently, is hydrogen, alkyl, cycloalkyl, heterocycloalkyl,C₁₋₃alkylenearyl, heteroaryl, or aryl, or both R groups are takentogether with the N to which they are attached to form a 4 to 8 memberedring.

The term “nitro” is defined as —NO₂.

The term “cyano” is defined as —CN.

The term “trifluoromethyl” is defined as —CF₃.

The term “trifluoromethoxy” is defined as —OCF₃.

The term “tBu” is defined as tertiary butyl, i.e. —C(CH₃)₃. The term“Boc” is defied as tert-butoxycarbonyl. The term “Bz” is defined asbenzyl.

As used herein, the term “aryl” refers to a monocyclic aromatic group,e.g., phenyl. Unless otherwise indicated, an aryl group can beunsubstituted or substituted with one or more, and in particular one tothree, groups independently selected from, for example, halo, alkyl,alkenyl, —OCF₃, —NO₂, —CN, —NC, —OH, alkoxy, amino, alkylamino, —CO₂H,—CO₂alkyl, alkynyl, cycloalkyl, nitro, sulfhydryl, imino, amido,phosphonate, phosphinate, silyl, alkylthio, sulfonyl, sulfonamide,aldehyde, heterocycloalkyl, trifluoromethyl, aryl, and heteroaryl.Exemplary aryl groups include, but are not limited to, phenyl,chlorophenyl, amidophenyl, aminophenyl, methylphenyl, methoxyphenyl,trifluoromethylphenyl, nitrophenyl, 2,4-methoxychlorophenyl, and thelike.

The term “C₁₋₆alkylenearylene” means

and serves to connect two other groups.

The term “C₂₋₆alkenylenearyleneC₁₋₄alkylene” means

and serves to connect two other groups.

As used herein, the term “heteroaryl” refers to a monocyclic ring systemcontaining at least one nitrogen, oxygen, or sulfur atom in an aromaticring. Unless otherwise indicated, a heteroaryl group can beunsubstituted or substituted with one or more, and in particular one tothree, substituents selected from, for example, halo, alkyl, alkenyl,—OCF₃, —NO₂, —CN, —NC, —OH, alkoxy, amino, alkylamino, —CO₂H, —CO₂alkyl,alkynyl, cycloalkyl, nitro, sulfhydryl, imino, amido, phosphonate,phosphinate, silyl, alkylthio, sulfonyl, sulfonamide, aldehyde,heterocycloalkyl, trifluoromethyl, aryl, and heteroaryl. Examples ofheteroaryl groups include, but are not limited to, thienyl, furyl,oxazolyl, thiophenyl, triazolyl, isothiazolyl, isoxazolyl, imidazolyl,pyrimidinyl, thiazolyl, thiadiazolyl, pyridinyl, pyridazinyl, pyrazolyl,pyrazinyl, tetrazolyl, oxazolyl, pyrrolyl, and triazinyl.

In some embodiments, the A group is —C(═O)NH-heteroaryl or—NHC(═O)heteroaryl, wherein the heteroaryl group is

either optionally substituted with an aryl, e.g., phenyl, group. In turnthe phenyl group also can be substituted, for example with —NHBoc,—NHC(═O)OC₂H₅, —NH₂, —NHC(═O)-t-butyl, —NHBz, or —NHC(═O)cyclohexyl.

In another embodiment, the A group is

wherein the heteroaryl group is

and the aryl group is phenyl, optionally substituted with halo, e.g.,chloro.

Additionally, salts, prodrugs, hydrates, isotopically labeled,fluorescently labeled and any other therapeutically or diagnosticallyrelevant derivations of the present HDACIs also are included in thepresent invention and can be used in the methods disclosed herein. Thepresent invention further includes all possible stereoisomers andgeometric isomers of the present compounds. The present inventionincludes both racemic compounds and optically active isomers. When apresent HDACI is desired as a single enantiomer, it can be obtainedeither by resolution of the final product or by stereospecific synthesisfrom either isomerically pure starting material or use of a chiralauxiliary reagent, for example, see Z. Ma et al., Tetrahedron:Asymmetry, 8(6), pages 883-888 (1997). Resolution of the final product,an intermediate, or a starting material can be achieved by any suitablemethod known in the art. Additionally, in situations where tautomers ofa present compound is possible, the present invention is intended toinclude all tautomeric forms of the compounds.

Prodrugs of the present compounds also are included in the presentinvention. It is well established that a prodrug approach, wherein acompound is derivatized into a form suitable for formulation and/oradministration, then released as a drug in vivo, has been successfullyemployed to transiently (e.g., bioreversibly) alter the physicochemicalproperties of the compound (see, H. Bundgaard, Ed., “Design ofProdrugs,” Elsevier, Amsterdam, (1985); R. B. Silverman, “The OrganicChemistry of Drug Design and Drug Action,” Academic Press, San Diego,chapter 8, (1992); K. M. Hillgren et al., Med. Res. Rev., 15, 83(1995)). Specific prodrugs of HDACIs are discussed in WO 2008/055068,incorporated in its entirety herein by reference.

Compounds of the present invention can contain one or more functionalgroups. The functional groups, if desired or necessary, can be modifiedto provide a prodrug. Suitable prodrugs include, for example, acidderivatives, such as amides and esters. It also is appreciated by thoseskilled in the art that N-oxides can be used as a prodrug.

Compounds of the invention can exist as salts. Pharmaceuticallyacceptable salts of the present HDACIs often are preferred in themethods of the invention. As used herein, the term “pharmaceuticallyacceptable salts” refers to salts or zwitterionic forms of the presentcompounds. Salts of the present compounds can be prepared during thefinal isolation and purification of the compounds or separately byreacting the compound with an acid having a suitable cation. Thepharmaceutically acceptable salts of the present compounds can be acidaddition salts formed with pharmaceutically acceptable acids. Examplesof acids which can be employed to form pharmaceutically acceptable saltsinclude inorganic acids such as nitric, boric, hydrochloric,hydrobromic, sulfuric, and phosphoric, and organic acids such as oxalic,maleic, succinic, tartaric, and citric. Nonlimiting examples of salts ofcompounds of the invention include, but are not limited to, thehydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate,2-hydroxyethansulfonate, phosphate, hydrogen phosphate, acetate,adipate, alginate, aspartate, benzoate, bisulfate, butyrate, camphorate,camphorsulfonate, digluconate, glycerolphosphate, hemisulfate,heptanoate, hexanoate, formate, succinate, fumarate, maleate, ascorbate,isethionate, salicylate, methanesulfonate, mesitylenesulfonate,naphthylenesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate,pamoate, pectinate, persulfate, 3-phenylproprionate, picrate, pivalate,propionate, trichloroacetate, trifluoroacetate, phosphate, glutamate,bicarbonate, paratoluenesulfonate, undecanoate, lactate, citrate,tartrate, gluconate, methanesulfonate, ethanedisulfonate, benzenesulphonate, and p-toluenesulfonate salts. In addition, available aminogroups present in the compounds of the invention can be quaternized withmethyl, ethyl, propyl, and butyl chlorides, bromides, and iodides;dimethyl, diethyl, dibutyl, and diamyl sulfates; decyl, lauryl,myristyl, and stearyl chlorides, bromides, and iodides; and benzyl andphenethyl bromides. In light of the foregoing, any reference tocompounds of the present invention appearing herein is intended toinclude the present compounds as well as pharmaceutically acceptablesalts, hydrates, or prodrugs thereof.

The present compounds also can be conjugated or linked to auxiliarymoieties that promote a beneficial property of the compound in a methodof therapeutic use. Such conjugates can enhance delivery of thecompounds to a particular anatomical site or region of interest (e.g., atumor), enable sustained therapeutic concentrations of the compounds intarget cells, alter pharmacokinetic and pharmacodynamic properties ofthe compounds, and/or improve the therapeutic index or safety profile ofthe compounds. Suitable auxiliary moieties include, for example, aminoacids, oligopeptides, or polypeptides, e.g., antibodies, such asmonoclonal antibodies and other engineered antibodies; and natural orsynthetic ligands to receptors in target cells or tissues. Othersuitable auxiliaries include fatty acid or lipid moieties that promotebiodistribution and/or uptake of the compound by target cells (see,e.g., Bradley et al., Clin. Cancer Res. (2001) 7:3229).

Specific compounds of the present invention include, but are not limitedto,

Synthetic Methods

The following synthetic schemes are representative of the reactions usedto synthesize the present HDACIs. Modifications and alternate schemes toprepare HDACIs of the invention are readily within the capabilities ofpersons skilled in the art.

It should be understood that protecting groups can be utilized inaccordance with general principles of synthetic organic chemistry toprovide compounds of the present invention. Protecting group-formingreagents are well known to persons skilled in the art, for example, seeT. W. Greene et al., “Protective Groups in Organic Synthesis, ThirdEdition,” John Wiley and Sons, Inc., NY, N.Y. (1999). These protectinggroups are removed when necessary by appropriate basic, acidic, orhydrogenolytic conditions known to persons skilled in the art.Accordingly, compounds of the present invention not specificallyexemplified herein can be prepared by persons skilled in the art.

General Synthetic Schemes

Analytical Data.

¹H NMR and ¹³C NMR spectra were recorded on Bruker spectrometer at 400MHz and 100 MHz respectively with TMS as an internal standard. Standardabbreviations indicating multiplicity were used as follows: s=singlet,b.s=broad singlet, d=doublet, t=triplet, q=quadruplet, and m=multiplet.HRMS experiments were performed on LTO-FTICR or Shimadzu IT-TOF MassSpectrometers. TLC was performed with Merck 250-mm 60F₂₅₄ silica gelplates. Preparative TLC was performed with Analtech 1000-mm silica gelGF plates. Column chromatography was performed using Merck silica gel(40-60 mesh). Analytical HPLC was carried out on an Ace 3AQ column(100×4.6 mm), with a Shimadzu 10 VP Series HPLC with a diode arraydetector; flow rate=2.0 mL/min; from 10% acetonitrile in water to 50% in10 min and to 100% acetonitrile in 5 min with 0.05% TFA (Method A), orfrom 30% acetonitrile in water to 100% of acetonitrile in 15 min with0.05% TFA (Method B) or from 30% acetonitrile in water to 100% ofacetonitrile in 30 min with 0.05% TFA (Method C), a column Ace AQ5(250×10 mm) was used with this last method.

5-[2-(4-Phenyl-thiazol-2-ylcarbamoyl)-ethyl]-isoxazole-3-carboxylic acidhydroxyl amide (Ex. 1): ¹H NMR (DMSO-d₆, 400 MHz) δ 2.92 (t, J=7.2 Hz,2H), 3.16 (t, J=7.2 Hz, 3H), 6.58 (s, 1H), 7.31-7.45 (m, 3H), 7.90 (d,J=7.4 Hz, 2H), 9.35 (s, 1H), 11.49 (s, 1H), 12.38 (s, 1H); ¹³C NMR(DMSO-d₆, 100 MHz) 21.8, 31.1, 32.5, 191.1, 108.4, 126.0, 128.2, 129.1,134.7, 149.2, 156.6, 157.9, 158.1, 170.1, 173.8; FAB-HRMS calcd forC₁₆H₁₄N₄O₄S [M+H]⁺: 359.0808; found: 359.0820.

5-[3-(4-Phenyl-thiazol-2-ylcarbamoyl)-propyl]-isoxazole-3-carboxylicacid hydroxyamide (Ex. 2): ¹H NMR (DMSO-d₆, 400 MHz) δ 2.02 (m, 2H),2.54 (t, J=7.4 Hz, 2H), 2.86 (t, J=7.4 Hz, 3H), 3.30 (b.s, 1H), 6.58 (s,1H), 7.30-7.45 (m, 3H), 7.88 (d, J=7.4 Hz, 2H), 9.35 (s, 1H), 12.28 (s,1H); ¹³C NMR (DMSO-d₆, 100 MHz) (partially degraded during overnightexperiment) 22.8, 25.7, 34.3, 102.3, 108.3, 124.6, 126.0, 128.1, 129.1,134.7, 149.1, 158.2, 171.2, 171.5; FAB-HRMS calcd for C₁₇H₁₆N₄O₄S[M+H]⁺: 373.0965; found: 373.0978.

(4-{2-[4-(3-Hydroxycarbamoyl-isoxazol-5-yl)-butyrylamino]-thiazol-4-yl}-phenyl)-carbamicacid tert-butyl ester (Ex. 3): ¹H NMR (DMSO-d₆, 400 MHz) δ: 1.41 (s,9H), 1.93 (m, 2H), 2.44 (m, 2H), 2.79 (m, 2H), 6.54 (s, 1H), 7.37-7.44(m 3H), 7.89 (d, J=5.1 Hz, 2H), 9.37 (s, 1H), 11.40 (b.s, 1H), 12.19 (s,1H); ¹³C NMR (DMSO-d₆, 100 MHz) δ: 22.8, 25.6, 28.5, 34.3, 79.6, 101.1,106.6, 118.5, 126.4, 128.7, 139.5, 149.1, 153.1, 156.6, 157.8, 158.1,171.1, 174.3; FAB-HRMS calcd for C₂₂H₂₅N₅O₆S [M+H]⁺: 488.1598; found:488.1618.

(4-{2-[4-(3-Hydroxycarbamoyl-isoxazol-5-yl)-butyrylamino]-thiazol-4-yl}-phenyl)-carbamicacid ethyl ester (Ex. 4): ¹H NMR (DMSO-d₆, 400 MHz) δ: 1.25 (t, J=7.1Hz, 3H), 2.02 (m, 2H), 2.54 (m, 2H), 2.86 (m, 2H), 4.14 (d, J=7.1 Hz,3H), 6.61 (s, 1H), 7.46 (s, 1H), 7.50 (d, J=7.1 Hz, 2H), 7.78 (d, J=7.1,2H), 9.72 (s, 1H), 11.48 (s, 1H), 12.27 (s, 1H); ¹³C NMR (DMSO-d₆, 100MHz) δ: 14.9, 22.8, 25.7, 31.1, 34.2, 60.6, 101.1, 106.8, 118.5, 126.5,129.0, 139.2, 149.0, 153.9, 157.9, 158.1, 171.1, 174.3; FAB-HRMS calcdfor C₂₀H₂₁N₅O₆S [M+H]⁺: 460.1285; found: 460.1300.

(3-{2-[3-(3-Hydroxycarbamoyl-isoxazol-5-yl)-propionylamino]-thiazol-4-yl}-phenyl)-carbamicacid ethyl ester (Ex. 5): The standard procedure used to generatehydroxamic acid (0.016 g, 51%). ¹H NMR (DMSO-d₆, 400 MHz) □ 1.30 (t,J=7.0 Hz, 3H), 2.95 (t, J=7.3 Hz, 2H), 3.25 (t, J=7.3 Hz, 2H), 4.19 (q,J=7.0 Hz, 2H), 6.60 (s, 1H), 7.28-7.32 (m, 3H), 7.55 (d, J=7.0 Hz, 1H),8.07 (s, 1H), 9.27 (s, 1H); ¹³C NMR (DMSO-d₆, 100 MHz) 14.9, 21.9, 32.5,60.6, 102.3, 108.5, 116.1, 118.2, 120.2, 129.4, 135.2, 140.0, 149.3,153.9, 157.4, 158.0, 161.3, 170.1, 174.6; FAB-HRMS calcd for C₁₉H₁₉N₅O₆S[M+H]⁺: 446.1128; found: 446.1141.

(4-{3-[2-(3-Hydroxycarbamoyl-isoxazol-5-yl)-ethylcarbamoyl]-isoxazol-5-yl}-phenyl)-carbamicacid tert-butyl ester (Ex. 6): ¹H NMR (DMSO-d₆, 400 MHz) δ: 1.42 (s,9H), 3.04 (m, 2H), 3.52 (m, 2H), 6.58 (s, 1H), 7.11 (s, 1H), 7.56 (d,J=8.4 Hz, 2H), 7.65 (d, J=8.4 Hz, 2H), 8.92 9 m, 1H), 9.28 (b.s, 1H),9.63 (s, 1H), 11.4 (s, 1H); ¹³C NMR (DMSO-d₆, 100 MHz) δ: 26.4, 28.4,37.2, 80.0, 98.8, 101.6, 118.5, 120.3, 127.0, 142.3, 153.0, 156.6,157.8, 159.1, 159.7, 170.9, 172.5; FAB-HRMS calcd for C₂₁H₂₃N₅O₇ [M+H]⁺:458.1670; found: 458.1669.

(3-{3-[2-(3-Hydroxycarbamoyl-isoxazol-5-yl)-ethylcarbamoyl]-isoxazol-5-yl}-phenyl)-carbamicacid tert-butyl ester (Ex. 7): ¹H NMR (DMSO-d₆, 400 MHz) δ: 1.42 (s,9H), 3.04 (m, 2H), 3.54 (m, 2H), 6.59 (s, 1H), 7.17 (s, 1H), 7.35 (d,J=7.8 Hz, 2H), 7.45 (m, 2H), 8.01 (s, 1H), 8.99 (t, J=5.5 Hz, 1H), 9.28(s, 1H), 9.54 (s, 1H), 11.42 (s, 1H); ¹³C NMR (DMSO-d₆, 100 MHz) δ:26.4, 28.5, 31.1, 37.2, 79.9, 100.1, 101.6, 114.9, 120.2, 120.8, 127.0,130.2, 140.8, 153.2, 156.6, 157.8, 158.9, 159.8, 170.9, 172.5; FAB-HRMScalcd for C₂₁H₂₃N₅O₇ [M+Na]⁺: 480.1490; found: 480.1505.

(4-{3-[2-(3-Hydroxycarbamoyl-isoxazol-5-yl)-ethylcarbamoyl]-isoxazol-5-yl}-phenyl)-carbamicacid ethyl ester (Ex. 8): ¹H NMR (DMSO-d₆, 400 MHz) δ: 1.17 (t, J=7.1Hz, 3H), 3.03 (m, 2H), 3.53 (m, 2H), 4.09 (q, J=6.5 Hz, 2H), 6.58 (s,1H), 7.12 (s, 1H), 7.54 (d, J=8.7 Hz, 2H), 7.76 (d, J=8.7 Hz, 2H), 8.93(t, J=5.7 Hz, 1H), 9.27 (s, 1H), 9.90 (s, 1H), 11.4 (s, 1H); ¹³C NMR(DMSO-d₆, 100 MHz) δ:14/8, 26.4, 37.2, 60.9, 98.9, 101.6, 118.6, 120.6,127.1, 142.0, 153.8, 156.6, 157.8, 159.1, 159.7, 170.9, 172.5; FAB-HRMScalcd for C₁₉H₁₉N₅O₇ [M+H]⁺: 430.1357; found: 430.1374.

5-(2-{[5-(3-Amino-phenyl)-isoxazole-3-carbonyl]-amino}-ethyl)-isoxazole-3-carboxylicacid hydroxyamide (Ex. 9): ¹H NMR (DMSO-d₆, 400 MHz) δ: 3.04 (m, 2H),3.53 (m, 2H), 5.36 (b.s, 2H), 6.58 (s, 1H), 6.65 (d, J=8.0 Hz, 1H), 6.98(m, 2H), 7.06 (s, 1H), 7.10 (m, 2H), 8.95 (t, J=5.4 Hz), 9.28 (s, 1H),11.42 (s, 1H); ¹³C NMR (DMSO-d₆, 100 MHz) δ: 26.4, 37.2, 99.4, 101.6,110.6, 113.9, 116.7, 127.1, 130.2, 149.6, 156.6, 157.8, 159.1, 159.6,171.7, 172.5; FAB-HRMS calcd for C₁₆H₁₅N₅O₅ [M+H]⁺: 358.1146; found:358.1156.

5-[2-({5-[3-(2,2-Dimethyl-propionylamino)-phenyl]-isoxazole-3-carbonyl}-amino)-ethyl]-isoxazole-3-carboxylicacid hydroxyamide (Ex. 10): ¹H NMR (DMSO-d₆, 400 MHz) δ: 1.18 (s, 9H),3.05 (m, 2H), 3.54 (m, 2H), 6.59 (s, 1H), 7.21 (s, 1H), 7.40 (t, J=7.8Hz, 1H), 7.56 (d, J=7.8 Hz, 1H), 7.76 (d, J=7.8 Hz, 1H), 8.19 (s, 1H),9.01 (t, J=5.0 Hz, 1H), 9.28 (s, 1H), 9.37 (s, 1H), 11.42 (s, 1H); ¹³CNMR (DMSO-d₆, 100 MHz) δ: 26.4, 27.5, 37.2, 100.1, 101.6, 117.2, 121.2,122.7, 126.8, 130.0, 140.6, 156.6, 157.8, 158.9, 159.8, 170.9, 172.5,177.2; FAB-HRMS calcd for C₂₁H₂₃N₅O₆ [M+H]⁺: 442.1721; found: 442.1738.

5-[2-({5-[3-Cyclohexanecarbonyl-amino)-phenyl]-isoxazole-3-carbonyl}-amino)-ethyl]-isoxazole-3-carboxylicacid hydroxyamide (Ex. 11): ¹H NMR (DMSO-d₆, 400 MHz) δ: 1.11-1.44 (m,5H), 1.58 (m, 2H), 1.74 (m, 2H), 2.28 (m, 1H), 3.05 (m, 2H), 3.54 (m,2H), 6.59 (s, 1H), 7.20 (m, 1H), 7.39 (t, J=7.6 Hz, 1H), 7.54 (d, J=7.6Hz, 1H), 7.60 (d, J=7.6 Hz, 1H), 8.18 (s, 1H), 9.00 (m, 1H), 9.98 (s,1H), 11.41 (s, 1H); ¹³C NMR (DMSO-d₆, 100 MHz) δ:25.6, 25.8, 26.4, 29.5,37.2, 45.3, 100.2, 101.6, 116.0, 121.0, 126.9, 130.2, 140.7, 156.6,157.8, 158.9, 159.8, 170.8, 172.5, 175.1; FAB-HRMS calcd for C₂₃H₂₅N₅O₆[M+H]⁺: 468.1877; found: 468.1873.

5-[2-({5-[3-Benzoylamino-amino)-phenyl]-isoxazole-3-carbonyl}-amino)-ethyl]-isoxazole-3-carboxylicacid hydroxyamide (Ex. 12).

¹H NMR (DMSO-d₆, 400 MHz) δ: 3.11 (t, J=6.6 Hz, 2H), 3.63 (dd, J=6.6 and12.7, 2H), 6.66 (s, 1H), 7.30 (s, 1H), 7.52-7.64 (m, 5H), 7.67 (d, J=7.9Hz, 1H), 7.94 (dd, J=1.2 and 6.6 Hz, 1H), 7.98 (m, 2H), 8.39 (m, 1H),9.08 (t, J=5.8 Hz, 1H), 9.35 (b.s, 1H), 10.48 (s, 1H), 11.49 (s, 1H);¹³C NMR (DMSO-d₆, 100 MHz) δ: 26.1, 36.9, 99.5, 99.9, 101.2, 117.0,121.4, 122.5, 126.6, 127.7, 128.5, 129.8, 131.8, 134.6, 140.0, 156.3,157.5, 158.6, 159.5, 165.8, 170.4, 172.2; FAB-HRMS calcd for C₂₃H₁₉N₅O₆[M+H]⁺: 462.1408; found: 462.1416.

5-(3-{[(4-Chloro-phenyl)-(1H-indazol-3-ylcarbamoyl)-methyl]-carbamoyl}-propyl)-isoxazole-3-carboxylicacid ethyl ester (Ex. 13): ¹H NMR (DMSO-d₆, 400 MHz) δ: 1.82 (m, 2H),2.26 (m, 2H), 2.73 (m, 1H), 5.67 (d, J=7.0 Hz, 1H), 6.49 (s, 1H), 6.95(t, J=7.0 Hz, 1H), 7.47 (t, J=7.0 Hz, 1H), 7.35 (d, J=7.0 Hz, 1H),7.41-7.54 (m, 5H), 8.67 (d, t, J=7.5 Hz, 1H), 9.28 (s, 1H), 10.72 (s,1H), 11.39 (s, 1H), 12.64 (s, 1H); ¹³C NMR (DMSO-d₆, 100 MHz) δ:23.1,15.2, 33.8, 56.9, 100.1, 109.7, 116.3, 120.0, 120.7, 126.8, 128.6,129.2, 134.0, 135.7, 139.1, 141.3, 156.9, 157.8, 169.8, 173.4, 174.5;FAB-HRMS calcd for C₂₃H₂₁ClN₆O₅ [M+H]⁺: 497.1334; found: 497.1356.

{3-[3-(4-Hydroxycarbamoyl-benzylcarbamoyl)-isoxazol-5-yl]-phenyl}-carbamicacid tert-butyl ester (Ex. 14): ¹H NMR (DMSO-d₆, 400 MHz) δ: 4.43 (d,J=5.9 Hz, 2H), 7.21 (s, 1H), 7.34 (m, 3H), 7.38 (m, 2H), 7.66 (d, J=8.1Hz, 2H), 8.03 (s, 1H), 9.38 (t, J=6.1 Hz, 1H), 9.55 (s, 1H), 11.12 (s,1H); ¹³C NMR (DMSO-d₆, 100 MHz) δ: 28.5, 42.5, 79.9, 100.2, 114.9,120.2, 120.7, 127.0, 127.4, 127.6, 130.2, 131.9, 140.8, 142.5, 153.2,159.0, 159.9, 164.5, 170.9; FAB-HRMS calcd for C₂₃H₂₄N₄O₆ [M+H]⁺:453.1774; found: 453.1781.

{4-[3-(4-Hydroxycarbamoyl-benzylcarbamoyl)-isoxazol-5-yl]-phenyl}-carbamicacid tert-butyl ester (Ex. 15): ¹H NMR (DMSO-d₆, 400 MHz) δ: 1.43 (s,9H), 4.43 (d, J=5.9 Hz, 2H), 7.15 (s, 1H), 7.31 (d, J=8.0 Hz, 2H), 7.55(d, J=8.6 Hz, 2H), 7.64 (d, J=8.0 Hz, 2H), 7.75 (d, J=8.6 Hz, 2H), 9.33(t, J=5.9 Hz, 1H), 9.64 (s, 1H), 11.11 (s, 1H); ¹³C NMR (DMSO-d₆, 100MHz) δ: 28.1, 30.7, 42.1, 79.7, 98.5, 99.5, 118.2, 120.0, 126.6, 127.0,127.2, 131.5, 142.0, 142.2, 152.6, 158.8, 159.4, 164.1, 170.6; FAB-HRMScalcd for C₂₃H₂₄N₄O₆ [M−H]⁻: 451.1623; found: 451.1639.

5-[4-(Cyclohexanecarbonyl-amino)-phenyl]-isoxazole-3-carboxylic acid4-hydroxycarbamoyl-benzylamide (Ex. 16): ¹H NMR (DMSO-d₆, 400 MHz) δ:1.11-1.76 (m, 10H), 2.32 (m, 1H), 4.44 (d, J=6.0 Hz, 2H), 7.16 (s, 1H),7.33 (d, J=8.1 Hz, 2H), 7.66 (d, J=8.1 Hz, 2H), 7.72 (d, J=8.6 Hz, 2H),7.80 (d, J=8.6 Hz, 2H), 8.94 (b.s, 1H), 9.35 (t, J=6.0 Hz, 1H), 10.05(s, 1H), 11.12 (s, 1H); ¹³C NMR (DMSO-d₆, 100 MHz) δ: 25.6, 25.7, 29.4,42.5, 45.3, 99.1, 119.6, 121.1, 127.0, 127.4, 127.5, 131.8, 142.1,142.5, 159.1, 159.8, 164.5, 170.8, 175.2; FAB-HRMS calcd for C₂₅H₂₆N₄O₆[M+H]⁺: 463.1976; found: 463.1991.

5-[3-(Cyclohexanecarbonyl-amino)-phenyl]-isoxazole-3-carboxylic acid4-hydroxycarbamoyl-benzylamide (Ex. 17): ¹H NMR (DMSO-d₆, 400 MHz) δ:1.18-1.84 (m, 10H), 2.33 (m, 1H), 4.51 (d, J=6.0 Hz, 2H), 7.30 (s, 1H),7.36 (d, J=8.2 Hz, 2H), 7.47 (t, J=7.9 Hz, 1H), 7.61 (d, J=7.9 Hz, 1H),7.71 (d, J=7.9 Hz, 1H), 7.73 (d, J=8.2 Hz, 2H), 8.26 (s, 1H), 9.00 (b.S,1H), 9.46 (t, J=6.0 Hz, 1H), 10.06 (s, 1H), 11.18 (s, 1H); ¹³C NMR(DMSO-d₆, 100 MHz) δ: 25.6, 25.8, 29.5, 42.5, 45.3, 100.2, 116.0, 121.0,121.6, 127.0, 127.4, 127.6, 130.2, 131.9, 140.7, 142.5, 159.0, 159.9,164.5, 170.8, 175.1; FAB-HRMS calcd for C₂₅H₂₆N₄O₆ [M+H]⁺: 463.1976;found: 463.1988.

5-Carbazol-9-ylmethyl-isoxazole-3-carboxylic acid4-hydroxycarbamoyl-benzylamide (Ex. 18): ¹H NMR (DMSO-d₆, 400 MHz) δ:4.32 (d, J=5.9 Hz, 2H), 5.88 (s, 2H), 7.18 (m, 4H), 7.42 (t, J=7.2 Hz,2H), 7.58 (d, J=8.2 Hz, 2H), 7.69 (d, J=8.2 Hz, 2H), 8.10 (d, J=7.2 Hz,2H), 8.92 (s, 1H), 9.21 (t, J=5.9 Hz, 1H), 11.07 (s, 1H); ¹³C NMR(DMSO-d₆, 100 MHz) δ: 37.8, 42.0, 102.4, 109.5, 119.6, 120.4, 122.5,126.0, 126.9, 127.1, 131.4, 139.7, 142.0, 158.3, 158.7, 164.1, 170.0;FAB-HRMS calcd for C₂₅H₂₀N₄O₄ [M+H]⁺: 441.1557; found: 441.1577.

5-[2-(4-Chloro-benzoylamino)-3-(1H-indol-3-yl)-propyl]-isoxazole-3-carboxylicacid hydroxyamide (Ex. 19): ¹H NMR (DMSO-d₆, 100 MHz) δ 2.91-3.16 (m,6H), 4.50 (m, 1H), 6.05 (s, 1H), 6.94 (t, J=5.0 Hz, 1H), 7.05 (t, J=5.0Hz, 1H), 7.18 (s, 1H), 7.32 (d, J=5.0 Hz, 2H), 7.52 (d, J=8.5 Hz, 2H),7.58 (d, J=5.0 Hz, 1H), 7.81 (d, J=8.5 Hz, 2H), 8.53 (d, J=4.0 Hz, 1H),10.85 (s, 1H); ¹³C NMR (DMSO-d₆, 100 MHz); FAB-HRMS calcd forC₂₂H₁₉ClN₅O₄ [M+H]⁺: 439.1167; found: 439.1179.

5-[3-(1H-Indol-3-yl)-2-(2,2,2-trifluoro-acetylamino)-propyl]-isoxazole-3-carboxylicacid hydroxyamide (Ex. 20): Standard procedure was used for preparation.The crude mixture was purified by HPLC (method A) to give hydroxamicacid as a white solid (0.021 g, 35%). NMR (DMSO-d₆, 100 MHz) δ2.93 (d,J=6.4 Hz, 2H), 3.01-3.14 (m, 2H), 6.44 (s, 1H), 6.92 (t, J=7.4 Hz, 1H),7.00 (t, J=7.4 Hz, 1H), 7.09 (s, 1H), 7.26 (d, J=7.4 Hz, 1H), 7.47 (d,J=7.4 Hz, 1H), 9.26 (s, 1H), 9.39 (d, J=8.3 Hz, 1H), 10.82 (s, 1H),11.41 (s, 1H); ¹³C NMR (DMSO-d₆, 100 MHz) δ: 29.8, 30.7, 50.1, 102.1,110.2, 111.9, 118.5, 118.8, 121.4, 124.0, 127.6, 136.5, 156.5, 157.7,171.8; FAB-HRMS calcd for C₁₇H₁₅F₃N₄O₄ [M+H]⁺: 397.1118; found:397.1136.

5-[2-(3,4-Dimethoxy-benzoylamino)-3-(1H-indol-3-yl)-propyl]-isoxazole-3-carboxylicacid hydroxyamide (Ex. 21): ¹H NMR (DMSO-d₆, 400 MHz) δ: 2.88-3.12 (m,4H), 3.71 (s, 3H), 3.72 (s, 3H), 4.50 (m, 1H), 6.44 (s, 1H), 6.91 (m,2H), 7.00 (t, J=7.5 Hz, 1H), 7.13 (d, J=2.0 Hz, 1H), 7.34 (m, 2H), 7.54(d, J=8.3 Hz, 1H), 8.22 (d, J=8.4 Hz, 1H), 9.24 (b.s, 1H), 10.78 (s,1H), 11.38 (s, 1H); ¹³C NMR (DMSO-d₆, 100 MHz) δ: 30.6, 31.1, 49.3,55.9, 56.0, 101.8, 111.13, 111.18, 111.24, 111.8, 118.7, 120.7, 121.3,124.0, 127.2, 127.8, 136.6, 148.5, 151.6, 156.6, 157.7, 166.0, 172.7;FAB-HRMS calcd for C₂₄H₂₄N₄O₆ [M+H]⁺: 465.1768; found: 465.1782.

The effectiveness, or potency, of a present HDACI with respect toinhibiting the activity of an HDAC is measured by an IC₅₀ value. Thequantitative IC₅₀ value indicates the concentration of a particularcompound that is needed to inhibit the activity of an enzyme by 50% invitro. Stated alternatively, the IC₅₀ value is the half maximal (50%)inhibitory concentration of a compound tested using a specific enzyme,e.g., HDAC, of interest. The smaller the IC₅₀ value, the more potent theinhibiting action of the compound because a lower concentration of thecompound is needed to inhibit enzyme activity by 50%.

In preferred embodiments, a present HDACI inhibits HDAC enzymaticactivity by about at least 50%, preferably at least about 75%, at least90%, at least 95%, or at least 99%.

Compounds of the present invention were tested for their potency toinhibit HDAC 1, 2, 3, 6, and 10. In some embodiments, present compoundsalso were tested against HDAC 4, 5, 7, 8 and 9. The tested compoundsshowed a range of IC₅₀ values vs. HDAC6 of less than 1 nM to no greaterthan 230 nM, and a range of IC₅₀ values vs. HDAC1 of about 200 nM togreater than 75 μM. Therefore, in some embodiments, a present HDACI is aselective HDAC6 inhibitor which, because of a low affinity for otherHDAC isozymes, e.g., HDAC1, give rise to fewer side effects thancompounds that are non-selective HDAC inhibitors.

In some embodiments, the present HDACIs interact with and reduce theactivity of all histone deacetylases in in vitro assay. In somepreferred embodiments, the present HDACIs interact with and reduce theactivity of fewer than all histone deacetylases in the in vitro assay.In certain preferred embodiments, the present HDACIs interact with andreduce the activity of one histone deacetylase (e.g., HDAC6), but do notsubstantially interact with or reduce the activities of other histonedeacetylases (e.g., HDAC1, HDAC2, HDAC3 HDAC4, HDAC5, HDAC7, HDAC8,HDAC9, HDAC10, and HDAC11).

The present invention therefore provides HDACIs for the treatment of avariety of diseases and conditions wherein inhibition of HDAC has abeneficial effect. Preferably, a present HDACI is selective for HDAC6over the other HDAC isozymes by a factor of at least 4, at least 10, atleast 20, at least 50, at least 100, at least 500, at least 1000, atleast 2000, at least 3000, and preferably up to about 7000. For example,in various embodiments, a present HDACI exhibits an IC₅₀ value versusHDAC6 that is about 350 or about 7000 times less than the IC₅₀ value vs.HDAC1, i.e., a selectivity ratio (HDAC1 IC₅₀/HDAC6 IC₅₀) of about 350 orabout 7000.

Other assays also showed selectivity of a present HDACI for HDAC6 overHDAC1, 2, 3, and 10 of about 1000 and 10000.

The IC₅₀ values for compounds of structural formula (I) vs. HDAC1 andHDAC6 were determined as follows:

The HDAC1, 2, 6, and 10 assays used isolated recombinant human protein;HDAC3/NcoR2 complex was used for the HDAC3 assay. Substrate for HDAC1,2, 3, 6, and 10 assays is a fluorogenic peptide from p53 residues379-382 (RHKKAc). Compounds were dissolved in DMSO and tested in 10-doseIC₅₀ mode with 3-fold serial dilution starting at 30 μM. ControlCompound Trichostatin A (TSA) was tested in a 10-dose IC₅₀ with 3-foldserial dilution starting at 5 μM. IC₅₀ values were extracted bycurve-fitting the dose/response slopes. Assays were performed induplicate and IC₅₀ values are an average of data from both experiments.

Materials

Human HDAC1 (GenBank Accession No. NM_004964): Full length withC-terminal GST tag, MW=79.9 kDa, expressed by baculovirus expressionsystem in 519 cells. Enzyme is in 50 mM Tris-HCl, pH 8.0, 138 mM NaCl,20 mM glutathione, and 10% glycerol, and stable for >6 months at −80° C.Purity is >10% by SDS-PAGE. Specific Activity is 20 U/μg, where one U=1pmol/min under assay condition of 25 mM Tris/Cl, pH8.0, 137 mM NaCl, 2.7mM KCl, 1 mM MgCl₂, 0.1 mg/ml BSA, 100 μM HDAC substrate, and 13.2 ng/μlHDAC1, incubation for 30 min at 30° C.

Human HDAC6 (GenBank Accession No. BC069243): Full length withN-terminal GST tag, MW=159 kDa, expressed by baculovirus expressionsystem in Sf9 cells. Enzyme is in 50 mM Tris-HCl, pH 8.0, 138 mM NaCl,20 mM glutathione, and 10% glycerol, and stable for >6 months at −80° C.Purity is >90% by SDS-PAGE. Specific Activity is 50 U/μg, where one U=1pmol/min under assay condition of 25 mM Tris/Cl, pH8.0, 137 mM NaCl, 2.7mM KCl, 1 mM MgCl₂, and 0.1 mg/ml BSA, 30 μM HDAC substrate, and 5 ng/μlHDAC6, incubation for 60 min at 30° C.

Substrate for HDAC1 and HDAC6: Acetylated peptide substrate for HDAC,based on residues 379-382 of p53 (Arg-His-Lys-Lys(Ac)), a site ofregulatory acetylation by the p300 and CBP acetyltransferases (lysines381, 382)1-6, is the best for HDAC from among a panel of substratespatterned on p53, histone H3 and histone H4 acetylation sites7.

References: W. Gu et al., Cell (1997) 90 595; K. Sakaguchi et al., GenesDev., (1998) 12 2831; L. Liu et al., Mol. Cell. Biol., (1999) 19 1202;A. Ito et al., EMBO J. (2001) 20 1331; N. A. Barley et al., Mol. Cell,(2001) 8 1243; and A. Ito et al., EMBO 1, (2002) 21 6236.

Reaction Buffer: 50 mM Tris-HCl, pH 8.0, 137 mM NaCl, 2.7 mM KCl, 1 mMMgCl₂, 1 mg/ml BSA.

Assay Conditions

HDAC1: 75 nM HDAC1 and 50 μM HDAC substrate are in the reaction bufferand 1% DMSO final. Incubate for 2 hours at 30° C.

HDAC6: 12.6 nM HDAC6 and 50 μM HDAC substrate are in the reaction bufferand 1% DMSO final. Incubate for 2 hours at 30° C.

IC₅₀ Calculations

All IC₅₀ values are automatically calculated using the GraphPad Prismversion 5 and Equation of Sigmoidal dose-response (variable slope):

Y=Bottom+(Top−Bottom)/(1+10{circumflex over ( )}((LogEC50−X)*HillSlope)), where X is the logarithm of concentration, Y is theresponse, Y starts at Bottom and goes to Top with a sigmoid shape. Inmost cases, “Bottom” is set 0, and “Top” is set “less than 120%”. Thisis identical to the “four parameter logistic equation”. IC₅₀ curves alsoare drawn using the GraphPad Prism, and IC₅₀ values and Hill slopes areprovided.

HDAC activity assays: HDAC assay is performed usingfluorescently-labeled acetylated substrate, which comprises anacetylated lysine side chain. After incubation with HDAC, deacetylationof the substrate sensitizes the substrate such that, in a second step,treatment with the detection enzyme produces a fluorophore. HDACs 1 and6 were expressed as full length fusion proteins. Purified proteins wereincubated with 50 μM fluorescently-labeled acetylated peptide substrateand test compound for 2 hours at RT in HDAC assay buffer containing 50mM Tris-HCl (pH 8.0), 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl₂, 1% DMSO, and1% BSA.

Reactions were terminated by the addition of the Developer after 2hours, and the development of fluorescence signal, which was relative tothe amount of deacetylated peptide, was monitored by time-coursemeasurement of EnVision (PerkinElmer). The HDAC activity was estimatedfrom the slope of time-course measurement of the fluorescence intensity.The slope of no-enzyme control (substrate alone) was served asbackground, and % Enzyme activity was calculated usingbackground-subtracted slope of no inhibitor control (DMSO) as 100%activity.

To date, HDACIs have demonstrated a relatively non-specific inhibitionof various HDAC isozymes. Most HDACI so far identified primarily inhibitHDAC 1, 2, 3, and 8, producing an antiproliferative phenotype which isuseful for oncology applications, but not for the many non-oncologyapplications of HDACIs. (K. B. Glaser et al, Biochemical and biophysicalresearch communications 2003, 310, 529-36.) The potential toxicitiesassociated with the inhibition of certain HDAC isozymes can lead toadditional difficulties for the clinical development of pan-HDAC, i.e.,nonselective HDAC, inhibitors. Because the network of cellular effectsmediated by acetylation is so vast and because inhibition of some HDACisozymes may lead to undesirable side effects, HDAC isozyme selectiveinhibitors hold a greater therapeutic promise than their nonselectivecounterparts.

As illustrated below, many HDACIs of the present invention exhibitselective inhibition of HDAC6 compared to other HDAC isozymes.

HDAC isoform inhibition of new HDACIs. All compounds were tested againstboth Class I (1, 2, and 3) and Class II (6 and 10) HDACs, and their IC₅₀values are shown in Table 1. In the isoxazole series, compounds 6, 7,11, and 12 are the best in terms of potency to inhibit HDAC6 andselectivity over all other isoforms tested (Table 1). The selectivityprofile of compound 6 was more closely evaluated (Table 2). In thebenzene series, the most potent HDAC6 inhibitors are the compounds 14,15, 17, and 18.

TABLE 1 In vitro HDAC inhibition assay results. HDAC isoform IC₅₀(nM)^(a) Compound Structure 1 2 3 6 10 Trichostatin A

3.0 6.4 7.3 0.7 8.9 1

307 1140 320 31 407 2

351 1220 934 81.8 854 3

401 1140 448 41.2 52.9 4

200 834 354 48.9 323 5

266 1100 107 3.3 271 6

16900 123000 22800 6.0 NI^(b) 7

54000 NI^(b) 98800 7.7 NI^(b) 8

3910 41300 5190 101 12500 9

6680 2360 1770 16.8 5290 10

76000 NI^(b) NI^(b) 21.2 40100 11

NI^(b) NI^(b) NI^(b) 6.7 NI^(b) 12

2930 10400 7050 4.4 4630 16

4580 11300 4050 221 6920 14

444 1380 789 0.64 2730 15

436 1900 135 0.33 3160 16

350 2490 215 5.4 204 17

212 4300 669 2.6 191 18

495 1370 479 0.6 22200 19

46600 NI^(b) 37800 430 NI^(b) 20

NI^(b) NI^(b) 41600 3050 NI^(b) 21

49900 NI^(b) 54100 721 NI^(b) ^(a)Performed by Reaction BiologyCorporation (http://www.reactionbiology.com) ^(b)No inhibition at aconcentration of 50 pM

TABLE 2 Selectivity of compound 6 Selectivity for Isoform IC₅₀ (nM)^(a)HDAC6 (fold) HDAC1 16900 2820 HDAC2 123000 20500 HDAC3 22800 3800 HDAC4130000 21600 HDAC5 34500 5750 HDAC6 6.0 — HDAC7 105000 17500 HDAC8 810135 HDAC9 12800 2130 HDAC10 >100000 >16000 ^(a)Performed by ReactionBiology Corporation

In Vitro growth inhibition of pancreatic cancer cell lines. Four of thepresent HDACIs were analyzed in the[3-(4,5-dimethylthiazol-2-yl)]-2,5-diphenyltetrazolium bromide (MTT)assay in four pancreatic cancer cell lines (Table 3). Each of compounds1, 2, and 4 were shown to be active against at least one cell line atlow-micromolar concentrations.

TABLE 3 In Vitro growth inhibition of pancreatic cancer cell lines bynew HDACIs IC₅₀ (μM) ^(a) Compound BxPC-3 Panc 04.03 Mia PaCa-2 PANC1  11  2 20.8 48.4 1.3 2.5  4 15.2 13.5 1.9 11.0 13 >50 >50 >50 >50 ^(a) Thepancreatic cancer cell lines were obtained from ATCC (Rockville, MD).[3-(4,5-Dimethylthiazol-2-yl)]-5-[3-(carboxymethoxy)phenyl]-2-(4-sulfophenyl)-2H-tetrazolium(MTS) assays were carried out according to the manufacturer's protocol.72 Hours post-treatment, cell viability was measured, and IC₅₀ valueswere determined.

Cell cycle and inhibition of alpha-tubulin deacetylation. Treatment ofpancreatic cancer cells with HDACIs leads to accumulation of acetylatedalpha-tubulin, a well-known target of HDAC6 (FIG. 1A).

This finding is consistent with cell cycle data, which indicate thattreatment of pancreatic cancer cells with a present HDACIs results in anaccumulation of cells in the G2/M phase of the cell cycle.Representative data are shown in FIG. 1B.

PANC1 Cell migration and effect on T and NK Cells. The effect of HDAC6inhibition on cell migration and cellular cytotoxicity was evaluated.Previous studies suggested that hyper-acetylation of microtubules wouldaffect both cell migration and MTOC polarization and cytotoxicity incytotoxic lymphocytes. To gain a better understanding of the utility andselectivity of the more selective HDAC6 inhibitors, pancreatic cancercells were treated with a series of HDAC6 inhibitors and measured cellmigration using the scratch-wound assay. Significantly, it was foundthat several compounds demonstrated a dramatic reduction in PANC1 cellmigration in this assay compared to diluent-treated control cells (FIG.2 and Table 4). Examples 6 and 9 had only a partial effect on cellmigration. Thus, these HDAC6-selective inhibitors could function well toblock metastasis. However, in contrast to the effect observed on cellmigration, none of the compounds tested affected CD8+ T cell or naturalkiller (NK) cell-mediated cytotoxicity, despite the ability of thecompounds to substantially increase the levels of acetyl-tubulin inthese cell lines. Thus, in contrast to what has been previously reportedusing SAHA, the present selective HDAC6 inhibitors do not affect T cellor NK cell-mediated immunity. This is an important finding because itindicates that the present compounds do not impair this aspect of immuneregulation, which is an important aspect in tumor eradication.

TABLE 4 Inhibition of cell migration ^(a) Compound 24 h 48 h Compound 24h 48 h DMSO − − Example 11 + − Example 6 + − Example 12 − − Example7 + + Example 14 − − Example 10 − − Example 18 + − Example 9 + − ^(a)Level of inhibition (high to low): +++, ++ or +; no inhibition: −Novel HDAC6 inhibitors have shorter linkers between the CAP region andthe zinc-chelating hydroxamate function, as well as rigidifide linkerwith incorporated an isoxazole or benzene ring. Among these, severalcompounds posess low-nanomolar to sub-nanomolar activity and high HDAC6selectivity. Selected compounds demonstrate low-micromolarantiproliferative activity against at least one pancreatic cancer cellline. In vitro treatment with HDACIs caused accumulation of the putativeHDAC6 targets, p21 and p27. Apoptosis was induced in Panc04.03 cells.Levels of the anti-apoptotic protein, XIAP and Bcl2, were reduced byvarious HDACIs. Treatment of pancreatic cancer cells with HDACIs lead toaccumulation of acetylated alpha-tubulin, which is in accordance withcell cycle data, indicating accumulation in the G2/M phase. HDAC6inhibition reduced cell migration in the scratch-wound assay, a model ofmetastasis, while not affecting CD8+ T cell or natural killer (NK)cell-mediated cytotoxicity, an important aspect in tumor eradication.HDACI treatment caused loss of the DNA damage checkpoint kinase Chk1,and thereby sensitized pancreatic cancer cells to gemcitabine, resultingin growth inhibition at lower combined doses of both drugs compared totreatment with each individual drug.Growth Inhibition of Pancreatic Cancer Cells by New HDACIs.

Some of the HDACIs listed in Table 5, showed antitumor activity whentested in two pancreatic cancer cell lines, BxPC3 and L3.6p1. Compounds1, 2, and 4 effectively induced apoptosis as determined by Hoechststaining and PARP cleavage, a marker of apoptosis, in HDACI-treatedL3.6pl pancreatic cancer cells 24 hours after the initiation oftreatment (FIG. 3 ).

TABLE 5 In vitro growth inhibition (GI) of pancreatic cancer cells bynew HDACIs. GI₅₀ (μM) ^(a) Compound BxPC3 L3.6pl 1 1.5 1.3 2 2.3 0.7 41.7 1.7 5 7.0 2.1 6 29 >50 7 >50 >50 8 >50 47 9 12 3.3 11 >50 >5012 >50 >50 14 7.8 >50 15 8.1 2 18 2.4 1.3 ^(a) Pancreatic cancer celllines BXTC3 and L3.6pl were obtained from American Type CultureCollection (ATCC, Manassas, VA). Relative number of viable cancer cellswas determined 72 hours post-treatment by measuring the optical densityusing[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt; MTS] cell proliferation assay kit (Promega, Madison, WI).GI50 value for each compound was calculated with a non-linear regressionmodel of standard slope using GraphPad. Prism 6.0 software.

The present HDACIs therefore demonstrate excellent HDAC6 potency andselectivity, often exhibiting an IC₅₀ of, <10 nM at HDAC6 and 1000- to7000-fold selectivity against HDAC1. For example, compound 6demonstrated an IC₅₀ of 7.7 nM at HDAC6 and 7012-fold selectivityagainst HDAC1.

The present compounds have been evaluated for their activity at HDAC6and their selectivity for HDAC6 over class 1 HDACs. Previously it wasshown that selective HDAC6 inhibitors are implicated in a variety ofdisease states including, but not limited to, arthritis, autoimmunedisorders, inflammatory disorders, cancer, neurological diseases such asRett syndrome, peripheral neuropathies such as CMT, stroke,hypertension, and diseases in which oxidative stress is a causativefactor or a result thereof. It also was shown that selective HDAC6inhibitors, when administered in combination with rapamycin, prolongedthe lifespan of mice with kidney xenografts. This model was used toevaluate the immunosuppressant properties of the present compounds andserve as a model of transplant rejection. Furthermore, it was previouslyshown that selective HDAC6 inhibitors confer neuroprotection in ratprimary cortical neuron models of oxidative stress. These studiesidentified selective HDAC6 inhibitors as non-toxic neuroprotectiveagents. The present compounds behave in a similar manner because theyalso are selective HDAC6 agents. The present compounds demonstrate aligand efficiency that renders them more drug-like in theirphysiochemical properties. In addition, the present compounds maintainthe potency and selectivity observed in prior HDACIs. The presentcompounds therefore are pharmaceutical candidates and research tools toidentify the specific functions of HDAC6.

In one embodiment, the present invention relates to a method of treatingan individual suffering from a disease or condition wherein inhibitionof HDACs provides a benefit comprising administering a therapeuticallyeffective amount of a present HDACI compound to an individual in needthereof.

The methods described herein relate to the use of a present HDACI and anoptional second therapeutic agent useful in the treatment of diseasesand conditions wherein inhibition of HDAC provides a benefit. Themethods of the present invention can be accomplished by administering apresent HDACI as the neat compound or as a pharmaceutical composition.Administration of a pharmaceutical composition, or a neat HDACI of thepresent invention, can be performed during or after the onset of thedisease or condition of interest. Typically, the pharmaceuticalcompositions are sterile, and contain no toxic, carcinogenic, ormutagenic compounds that would cause an adverse reaction whenadministered.

In many embodiments, a present HDACI is administered in conjunction witha second therapeutic agent useful in the treatment of a disease orcondition wherein inhibition of HDAC provides a benefit. The secondtherapeutic agent is different from the present HDACI. A present HDACIand the second therapeutic agent can be administered simultaneously orsequentially. In addition, a present HDACI and second therapeutic agentcan be administered from a single composition or two separatecompositions. A present HDACI and the second therapeutic agent can beadministered simultaneously or sequentially to achieve the desiredeffect.

The second therapeutic agent is administered in an amount to provide itsdesired therapeutic effect. The effective dosage range for each secondtherapeutic agent is known in the art, and the second therapeutic agentis administered to an individual in need thereof within such establishedranges.

The present invention therefore is directed to compositions and methodsof treating diseases or conditions wherein inhibition of HDAC provides abenefit. The present invention also is directed to pharmaceuticalcompositions comprising a present HDACI and an optional secondtherapeutic agent useful in the treatment of diseases and conditionswherein inhibition of HDAC provides a benefit. Further provided are kitscomprising a present HDACI and, optionally, a second therapeutic agentuseful in the treatment of diseases and conditions wherein inhibition ofHDAC provides a benefit, packaged separately or together, and an inserthaving instructions for using these active agents.

A present HDACI and the second therapeutic agent can be administeredtogether as a single-unit dose or separately as multi-unit doses,wherein the present HDACI is administered before the second therapeuticagent or vice versa. One or more dose of a present HDACI and/or one ormore dose of the second therapeutic agent can be administered. Thepresent HDACIs therefore can be used in conjunction with one or moresecond therapeutic agents, for example, but not limited to, anticanceragents.

Within the meaning of the present invention, the term “disease” or“condition” denotes disturbances and/or anomalies that as a rule areregarded as being pathological conditions or functions, and that canmanifest themselves in the form of particular signs, symptoms, and/ormalfunctions. As demonstrated below, a present HDACI is a potentinhibitor of HDAC and can be used in treating diseases and conditionswherein inhibition of HDAC provides a benefit, for example, cancer, aneurological disease, a neurodegenerative condition, traumatic braininjury, stroke, an inflammation, an autoimmune disease, autism, andmalaria.

In one preferred embodiment, the present invention provides methods fortreating cancer, including but not limited to killing a cancer cell orneoplastic cell; inhibiting the growth of a cancer cell or neoplasticcell; inhibiting the replication of a cancer cell or neoplastic cell; orameliorating a symptom thereof, said methods comprising administering toa subject in need thereof a therapeutically effective amount of apresent HDACI.

In one embodiment, the invention provides a method for treating cancercomprising administering to a subject in need thereof an amount of apresent HDACI or a pharmaceutically acceptable salt thereof sufficientto treat the cancer. A present HDACI can be used as the sole anticanceragent, or in combination with another anticancer treatment, e.g.,radiation, chemotherapy, and surgery.

In another embodiment, the invention provides a method for increasingthe sensitivity of a cancer cell to the cytotoxic effects ofradiotherapy and/or chemotherapy comprising contacting the cell with apresent HDACI or a pharmaceutically acceptable salt thereof in an amountsufficient to increase the sensitivity of the cell to the cytotoxiceffects of radiotherapy and/or chemotherapy.

In a further embodiment, the present invention provides a method fortreating cancer comprising: (a) administering to an individual in needthereof an amount of a present HDACI compound; and (b) administering tothe individual an amount of radiotherapy, chemotherapy, or both. Theamounts administered are each effective to treat cancer. In anotherembodiment, the amounts are together effective to treat cancer.

In another embodiment, the invention provides a method for treatingcancer, said method comprising administering to a subject in needthereof a pharmaceutical composition comprising an amount of a presentHDACI effective to treat cancer.

This combination therapy of the invention can be used accordingly in avariety of settings for the treatment of various cancers. In a specificembodiment, the individual in need of treatment has previously undergonetreatment for cancer. Such previous treatments include, but are notlimited to, prior chemotherapy, radiotherapy, surgery, or immunotherapy,such as cancer vaccines.

In another embodiment, the cancer being treated is a cancer which hasdemonstrated sensitivity to radiotherapy and/or chemotherapy or is knownto be responsive to radiotherapy and/or chemotherapy. Such cancersinclude, but are not limited to, non-Hodgkin's lymphoma, Hodgkin'sdisease, Ewing's sarcoma, testicular cancer, prostate cancer, ovariancancer, bladder cancer, larynx cancer, cervical cancer, nasopharynxcancer, breast cancer, colon cancer, pancreatic cancer, head and neckcancer, esophageal cancer, rectal cancer, small-cell lung cancer,non-small cell lung cancer, brain tumors, or other CNS neoplasms.

In still another embodiment, the cancer being treated has demonstratedresistance to radiotherapy and/or chemotherapy or is known to berefractory to radiotherapy and/or chemotherapy. A cancer is refractoryto a therapy when at least some significant portion of the cancer cellsare not killed or their cell division is not arrested in response totherapy. Such a determination can be made either in vivo or in vitro byany method known in the art for assaying the effectiveness of treatmenton cancer cells, using the art-accepted meanings of “refractory” in sucha context. In a specific embodiment, a cancer is refractory where thenumber of cancer cells has not been significantly reduced or hasincreased.

Other cancers that can be treated with the compounds and methods of theinvention include, but are not limited to, cancers and metastasesselected from the group consisting of solid tumors, including but notlimited to: fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma,osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma,Ewing's tumor, leiornyosarcoma, rhabdomyosarcoma, colon cancer,colorectal cancer, kidney cancer, pancreatic cancer, bone cancer, breastcancer, ovarian cancer, prostate cancer, esophageal cancer, stomachcancer, oral cancer, nasal cancer, throat cancer, squamous cellcarcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma,sebaceous gland carcinoma, papillary carcinoma, papillaryadenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogeniccarcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma,choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervicalcancer, uterine cancer, testicular cancer, small cell lung carcinoma,bladder carcinoma, lung cancer, epithelial carcinoma, glioma,glioblastoma multiforma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, meningioma, skin cancer, melanoma,neuroblastoma, and retinoblastoma; blood-borne cancers, including butnot limited to: acute lymphoblastic leukemia, acute lymphoblastic B-cellleukemia, acute lymphoblastic T-cell leukemia, acute myeloblasticleukemia, acute promyelocytic leukemia, acute monoblastic leukemia,acute erythroleukemic leukemia, acute megakaryoblastic leukemia, acutemyclomonocytic leukemia, acute nonlymphocyctic leukemia, acuteundifferentiated leukemia, chronic myclocytic leukemia, chroniclymphocytic leukemia, hairy cell leukemia, and multiple myeloma; acuteand chronic leukemias: lymphoblastic, myelogenous lymphocytic, andmyelocytic leukemias; lymphomas: Hodgkin's disease and non-Hodgkin'slymphoma; multiple myeloma; Waldenstrom's macroglobulinemia; heavy chaindisease; and polycythemia vera.

The present HDACIs can also be administered to prevent progression to aneoplastic or malignant state, including but not limited to the cancerslisted above. Such prophylactic use is indicated in conditions known orsuspected of preceding progression to neoplasia or cancer, inparticular, where non-neoplastic cell growth consisting of hyperplasia,metaplasia, or most particularly, dysplasia has occurred (for review ofsuch abnormal growth conditions, see Robbins and Angell, 1976, BasicPathology, 2d Ed., W.B. Saunders Co., Philadelphia, pp. 68-79).Hyperplasia is a form of controlled cell proliferation involving anincrease in cell number in a tissue or organ, without significantalteration in structure or function. For example, endometrialhyperplasia often precedes endometrial cancer and precancerous colonpolyps often transform into cancerous lesions. Metaplasia is a form ofcontrolled cell growth in which one type of adult or fullydifferentiated cell substitutes for another type of adult cell.Metaplasia can occur in epithelial or connective tissue cells. A typicalmetaplasia involves a somewhat disorderly metaplastic epithelium.Dysplasia is frequently a forerunner of cancer, and is found mainly inthe epithelia; it is the most disorderly form of non-neoplastic cellgrowth, involving a loss in individual cell uniformity and in thearchitectural orientation of cells. Dysplastic cells often haveabnormally large, deeply stained nuclei, and exhibit pleomorphism.Dysplasia characteristically occurs where chronic irritation orinflammation exists, and often is found in the cervix, respiratorypassages, oral cavity, and gall bladder.

Alternatively or in addition to the presence of abnormal cell growthcharacterized as hyperplasia, metaplasia, or dysplasia, the presence ofone or more characteristics of a transformed phenotype, or of amalignant phenotype, displayed in vivo or displayed in vitro by a cellsample from a subject, can indicate the desirability ofprophylactic/therapeutic administration of the composition of theinvention. Such characteristics of a transformed phenotype include, forexample, morphology changes, looser substratum attachment, loss ofcontact inhibition, loss of anchorage dependence, protease release,increased sugar transport, decreased serum requirement, expression offetal antigens, disappearance of the 250,000 dalton cell surfaceprotein.

In a specific embodiment, leukoplakia, a benign-appearing hyperplasticor dysplastic lesion of the epithelium, or Bowen's disease, a carcinomain situ, are pre-neoplastic lesions indicative of the desirability ofprophylactic intervention.

In another embodiment, fibrocystic disease (cystic hyperplasia, mammarydysplasia, particularly adenosis (benign epithelial hyperplasia)) isindicative of the desirability of prophylactic intervention.

The prophylactic use of the compounds and methods of the presentinvention are also indicated in some viral infections that may lead tocancer. For example, human papilloma virus can lead to cervical cancer(see, e.g., Hernandez-Avila et al., Archives of Medical Research (1997)28:265-271), Epstein-Barr virus (EBV) can lead to lymphoma (see, e.g.,Herrmann et al., J Pathol (2003) 199(2):140-5), hepatitis B or C viruscan lead to liver carcinoma (see, e.g., El-Serag, J Clin Gastroenterol(2002) 35(5 Suppl 2):S72-8), human T cell leukemia virus (HTLV)-I canlead to T-cell leukemia (see e.g., Mortreux et al., Leukemia (2003)17(1):26-38), human herpesvirus-8 infection can lead to Kaposi's sarcoma(see, e.g., Kadow et al., Curr Opin Investig Drugs (2002) 3(11):1574-9),and Human Immune deficiency Virus (HIV) infection contribute to cancerdevelopment as a consequence of immunodeficiency (see, e.g., Dal Maso etal., Lancet Oncol (2003) 4(2):110-9).

In other embodiments, a subject exhibiting one or more of the followingpredisposing factors for malignancy can be treated by administration ofthe present HDACIs and methods of the invention: a chromosomaltranslocation associated with a malignancy (e.g., the Philadelphiachromosome for chronic myelogenous leukemia, t(14;18) for follicularlymphoma, etc.), familial polyposis or Gardner's syndrome (possibleforerunners of colon cancer), benign monoclonal gammopathy (a possibleforerunner of multiple myeloma), a first degree kinship with personshaving a cancer or procancerous disease showing a Mendelian (genetic)inheritance pattern (e.g., familial polyposis of the colon, Gardner'ssyndrome, hereditary exostosis, polyendocrine adenomatosis, medullarythyroid carcinoma with amyloid production and pheochromocytoma,Peutz-Jeghers syndrome, neurofibromatosis of Von Recklinghausen,retinoblastoma, carotid body tumor, cutaneous melanocarcinoma,intraocular melanocarcinoma, xeroderma pigmentosum, ataxiatelangiectasia, Chediak-Higashi syndrome, albinism, Fanconi's aplasticanemia, and Bloom's syndrome; see Robbins and Angell, 1976, BasicPathology, 2d Ed., W.B. Saunders Co., Philadelphia, pp. 112-113) etc.),and exposure to carcinogens (e.g., smoking, and inhalation of orcontacting with certain chemicals).

In another specific embodiment, the present HDACIs and methods of theinvention are administered to a human subject to prevent progression ofbreast, colon, ovarian, or cervical cancer.

In one embodiment, the invention provides methods for treating cancercomprising (a) administering to an individual in need thereof an amountof a present HDACI; and (b) administering to the individual one or moreadditional anticancer treatment modality including, but not limited to,radiotherapy, chemotherapy, surgery or immunotherapy, such as a cancervaccine. In one embodiment, the administering of step (a) is prior tothe administering of step (b). In another embodiment, the administeringof step (a) is subsequent to the administering of step (b). In stillanother embodiment, the administering of step (a) is concurrent with theadministering of step (b).

In one embodiment, the additional anticancer treatment modality isradiotherapy and/or chemotherapy. In another embodiment, the additionalanticancer treatment modality is surgery.

In still another embodiment, the additional anticancer treatmentmodality is immunotherapy, such as cancer vaccines.

In one embodiment, a present HDACI or a pharmaceutically acceptable saltthereof is administered adjunctively with the additional anticancertreatment modality.

In a preferred embodiment, the additional anticancer treatment modalityis radiotherapy. In the methods of the present invention, anyradiotherapy protocol can be used depending upon the type of cancer tobe treated. Embodiments of the present invention employ electromagneticradiation of: gamma-radiation (10⁻²⁰ to 10⁻¹³ m), X-ray radiation (10⁻¹²to 10⁻⁹ m), ultraviolet light (10 nm to 400 nm), visible light (400 nmto 700 nm), infrared radiation (700 nm to 1 mm), and microwave radiation(1 mm to 30 cm).

For example, but not by way of limitation, X-ray radiation can beadministered; in particular, high-energy megavoltage (radiation ofgreater that 1 MeV energy) can be used for deep tumors, and electronbeam and orthovoltage X-ray radiation can be used for skin cancers.Gamma-ray emitting radioisotopes, such as radioactive isotopes ofradium, cobalt and other elements, can also be administered.Illustrative radiotherapy protocols useful in the present inventioninclude, but are not limited to, stereotactic methods where multiplesources of low dose radiation are simultaneously focused into a tissuevolume from multiple angles; “internal radiotherapy,” such asbrachytherapy, interstitial irradiation, and intracavitary irradiation,which involves the placement of radioactive implants directly in a tumoror other target tissue; intraoperative irradiation, in which a largedose of external radiation is directed at the target tissue which isexposed during surgery; and particle beam radiotherapy, which involvesthe use of fast-moving subatomic particles to treat localized cancers.

Many cancer treatment protocols currently employ radiosensitizersactivated by electromagnetic radiation, e.g., X-rays. Examples ofX-ray-activated radiosensitizers include, but are not limited to,metronidazole, misonidazole, desmethylmisonidazole, pimonidazole,etanidazole, nimorazole, mitomycin C, RSU 1069, SR 4233, E09, RB 6145,nicotinamide, 5-bromodeoxyuridine (BUdR), 5-iododeoxyuridine (IUdR),bromodeoxycytidine, fluorodeoxyuridine (FUdR), hydroxyurea, cis-platin,and therapeutically effective analogs and derivatives of the same.

Photodynamic therapy (PDT) of cancers employs visible light as theradiation activator of the sensitizing agent. Examples of photodynamicradiosensitizers include the following, but are not limited to:hematoporphyrin derivatives, PHOTOFRIN®, benzoporphyrin derivatives,NPe6, tin etioporphyrin (SnET2), pheoborbide-a, bacteriochlorophyll-a,naphthalocyanines, phthalocyanines, zinc phthalocyanine, andtherapeutically effective analogs and derivatives of the same.

Radiosensitizers can be administered in conjunction with atherapeutically effective amount of one or more compounds in addition toa present HDACI, such compounds including, but not limited to, compoundsthat promote the incorporation of radiosensitizers to the target cells,compounds that control the flow of therapeutics, nutrients, and/oroxygen to the target cells, chemotherapeutic agents that act on thetumor with or without additional radiation, or other therapeuticallyeffective compounds for treating cancer or other disease. Examples ofadditional therapeutic agents that can be used in conjunction withradiosensitizers include, but are not limited to, 5-fluorouracil (5-FU),leucovorin, oxygen, carbogen, red cell transfusions, perfluorocarbons(e.g., FLUOSOLW®-DA), 2,3-DPG, BW12C, calcium channel blockers,pentoxifylline, antiangiogenesis compounds, hydralazine, and L-BSO.

In a preferred embodiment, a present HDACI or a pharmaceuticallyacceptable salt thereof is administered prior to the administration ofradiotherapy and/or chemotherapy.

In another preferred embodiment, a present HDACI or a pharmaceuticallyacceptable salt thereof is administered adjunctively with radiotherapyand/or chemotherapy.

A present HDACI and additional treatment modalities can act additivelyor synergistically (i.e., the combination of a present HDACI or apharmaceutically acceptable salt thereof, and an additional anticancertreatment modality is more effective than their additive effects wheneach are administered alone). A synergistic combination permits the useof lower dosages of a present HDACI and/or the additional treatmentmodality and/or less frequent administration of a present HDACI and/oradditional treatment modality to a subject with cancer. The ability toutilize lower dosages of a present HDACI and/or an additional treatmentmodality and/or to administer a compound of the invention and theadditional treatment modality less frequently can reduce the toxicityassociated with the administration without reducing the efficacy of apresent HDACI and/or the additional treatment modality in the treatmentof cancer. In addition, a synergistic effect can result in the improvedefficacy of the treatment of cancer and/or the reduction of adverse orunwanted side effects associated with the administration of a presentHDACI and/or an additional anticancer treatment modality as monotherapy.

In one embodiment, the present HDACIs may act synergistically withradiotherapy when administered in doses typically employed when suchHDACIs are used alone for the treatment of cancer. In anotherembodiment, the present HDACIs may act synergistically with radiotherapywhen administered in doses that are less than doses typically employedwhen such HDACIs are used as monotherapy for the treatment of cancer.

In one embodiment, radiotherapy may act synergistically with a presentHDACI when administered in doses typically employed when radiotherapy isused as monotherapy for the treatment of cancer. In another embodiment,radiotherapy may act synergistically with a compound of the inventionwhen administered in doses that are less than doses typically employedwhen radiotherapy is used as monotherapy for the treatment of cancer.

The effectiveness of the HDACIs as HDAC inhibitors for sensitizingcancer cells to the effect of radiotherapy can be determined by the invitro and/or in vivo determination of post-treatment survival usingtechniques known in the art. In one embodiment, for in vitrodeterminations, exponentially growing cells can be exposed to knowndoses of radiation, and the survival of the cells monitored. Irradiatedcells are plated and cultured for about 14-about 21 days, and thecolonies are stained. The surviving fraction is the number of coloniesdivided by the plating efficiency of unirradiated cells. Graphing thesurviving fraction on a log scale versus the absorbed dose on a linearscale generates a survival curve. Survival curves generally show anexponential decrease in the fraction of surviving cells at higherradiation doses after an initial shoulder region in which the dose issublethal. A similar protocol can be used for chemical agents when usedin the combination therapies of the invention.

Inherent radiosensitivity of tumor cells and environmental influences,such as hypoxia and host immunity, can be further assessed by in vivostudies. The growth delay assay is commonly used. This assay measuresthe time interval required for a tumor exposed to radiation to regrow toa specified volume. The dose required to control about 50% of tumors isdetermined by the TCD₅₀ assay.

In vivo assay systems typically use transplantable solid tumor systemsin experimental subjects. Radiation survival parameters for normaltissues as well as for tumors can be assayed using in vivo methods knownin the art.

The present invention provides methods of treating cancers comprisingthe administration of an effective amount of a present HDACI inconjunction with recognized methods of surgery, radiotherapy, andchemotherapies, including, for example, chemical-based mimics ofradiotherapy whereby a synergistic enhancement of the effectiveness ofthe recognized therapy is achieved. The effectiveness of a treatment canbe measured in clinical studies or in model systems, such as a tumormodel in mice, or cell culture sensitivity assays.

The present invention provides combination therapies that result inimproved effectiveness and/or reduced toxicity. Accordingly, in oneaspect, the invention relates to the use of the present HDACIs asradiosensitizers in conjunction with radiotherapy.

When the combination therapy of the invention comprises administering apresent HDACI with one or more additional anticancer agents, the presentHDACI and the additional anticancer agents can be administeredconcurrently or sequentially to an individual. The agents can also becyclically administered. Cycling therapy involves the administration ofone or more anticancer agents for a period of time, followed by theadministration of one or more different anticancer agents for a periodof time and repeating this sequential administration, i.e., the cycle,in order to reduce the development of resistance to one or more of theanticancer agents of being administered, to avoid or reduce the sideeffects of one or more of the anticancer agents being administered,and/or to improve the efficacy of the treatment.

An additional anticancer agent may be administered over a series ofsessions; anyone or a combination of the additional anticancer agentslisted below may be administered.

The present invention includes methods for treating cancer comprisingadministering to an individual in need thereof a present HDACI and oneor more additional anticancer agents or pharmaceutically acceptablesalts thereof. A present HDACI and the additional anticancer agent canact additively or synergistically. Suitable anticancer agents include,but are not limited to, gemcitabine, capecitabine, methotrexate, taxol,taxotere, mereaptopurine, thioguanine, hydroxyurea, cyclophosphamide,ifosfamide, nitrosoureas, mitomycin, dacarbazine, procarbizine,etoposide, teniposide, campatheeins, bleomycin, doxorubicin, idarubicin,daunorubicin, dactinomycin, plicamycin, mitoxantrone, L-asparaginase,doxorubicin, epirubicin, 5-fluorouracil (5-FU), taxanes (such asdocetaxel and paclitaxel), leucovorin, levamisole, irinotecan,estramustine, etoposide, nitrogen mustards, BCNU, nitrosoureas (such ascarmustine and lomustine), platinum complexes (such as cisplatin,carboplatin and oxaliplatin), imatinib mesylate, hexamethylmelamine,topotecan, tyrosine kinase inhibitors, tyrphostins herbimycin A,genistein, erbstatin, and lavendustin A.

In one embodiment, the anti-cancer agent can be, but is not limited to,a drug selected from the group consisting of alkylating agents, nitrogenmustards, cyclophosphamide, trofosfamide, chlorambucil, nitrosoureas,carmustine (BCNU), lomustine (CCNU), alkyl sulphonates, busulfan,treosulfan, triazenes, plant alkaloids, vinca alkaloids (vineristine,vinblastine, vindesine, vinorelbine), taxoids, DNA topoisomcraseinhibitors, epipodophyllins, 9-aminocamptothecin, camptothecin,crisnatol, mitomycins, mitomycin C, anti-metabolites, anti-folates, DHFRinhibitors, trimetrexate, IMP dehydrogenase inhibitors, mycophenolicacid, tiazofurin, ribavirin, EICAR, ribonuclotide reductase inhibitors,hydroxyurea, deferoxamine, pyrimidine analogs, uracil analogs,floxuridine, doxifluridine, ratitrexed, cytosine analogs, cytarabine(ara C), cytosine arabinoside, fludarabine, purine analogs,mercaptopurine, thioguanine, DNA antimetabolites, 3-HP,2′-deoxy-5-fluorouridine, 5-HP, alpha-TGDR, aphidicolin glycinate,ara-C, 5-aza-2′-deoxycytidine, beta-TGDR, cyclocytidine, guanazole(inosine glycodialdehyde), macebecin II, pyrazoloimidazole, hormonaltherapies, receptor antagonists, anti-estrogen, tamoxifen, raloxifene,megestrol, LHRH agonists, goserelin, leuprolide acetate, anti-androgens,flutamide, bicalutamide, retinoids/deltoids, cis-retinoic acid, vitaminA derivative, all-trans retinoic acid (ATRA-IV), vitamin D3 analogs, El)1089, CB 1093, ICH 1060, photodynamic therapies, vertoporfin, BPD-MA,phthalocyanine, photosensitizer Pc4, demethoxy-hypocrellin A(2BA-2-DMHA), cytokines, interferon-a, interferon-I3, interferon-y,tumor necrosis factor, angiogenesis inhibitors, angiostatin (plasminogenfragment), antiangiogenic antithrombin UI, angiozyme, ABT-627, Bay12-9566, benefin, bevacizumab, BMS-275291, cartilage-derived inhibitor(CDI), CAI, CD59 complement fragment, CEP-7055, Col 3, combretastatinA-4, endostatin (collagen XVIII fragment), fibronectin fragment,Gro-beta, halofuginone, heparinases, heparin hexasaccharide fragment,HMV833, human chorionic gonadotropin (hCG), IM-862, interferon inducibleprotein (IP-10), interleukin-12, kringle 5 (plasminogen fragment),marimastat, metalloproteinase inhibitors (UMPs), 2-methoxyestradiol,MIMI 270 (CGS 27023A), MoAb IMC-I C11, neovastat, NM-3, panzem, P1-88,placental ribonuclease inhibitor, plasminogen activator inhibitor,platelet factor-4 (PF4), prinomastat, prolactin 161(D fragment,proliferin-related protein (PRP), PTK 787/ZK 222594, retinoids,solimastat, squalamine, SS 3304, SU 5416, SU 6668, SU 11248,tetrahydrocortisol-S, tetrathiomolybdate, thalidomide, thrombospondin-1(TSP-1), TNP-470, transforming growth factor-beta (TGF-11),vasculostatin, vasostatin (calreticulin fragment), ZD 6126, ZD 6474,famesyl transferase inhibitors (FTI), bisphosphonates, antimitoticagents, allocolchicine, halichondrin B, colchicine, colchicinederivative, dolstatin 10, maytansine, rhizoxin, thiocolchicine, tritylcysteine, isoprenylation inhibitors, dopaminergic neurotoxins,1-methyl-4-phenylpyridinium ion, cell cycle inhibitors, staurosporine,actinomycins, actinomycin D, dactinomycin, bleomycins, bleomycin A2,bleomycin B2, peplomycin, anthracycline, adriamycin, epirubicin,pirarnbicin, zorubicin, mitoxantrone, MDR inhibitors, verapamil,Ca²′ATPase inhibitors, and thapsigargin.

Other anti-cancer agents that may be used in the present inventioninclude, but are not limited to, acivicin; aclarubicin; acodazolehydrochloride; acronine; adozelesin; aldesleukin; altretamine;arnbomycin; ametantrone acetate; aminoglutethimide; amsacrine;anastrozole; anthramycin; asparaginase; asperlin; azacitidine; azetepa;azotomycin; batimastat; benzodepa; bicalutamide; bisantrenehydrochloride; bisnafide dimesylate; bizelcsin; bleomycin sulfate;brequinar sodium; bropirimine; busul fan; cactinomycin; calusterone;caracemide; carbetimer; carmustine; carubicin hydrochloride; carzelesin;cedefingol; chlorambucil; cirolemycin; cisplatin; cladribine; crisnatolmesylate; cyclophosphamide; cytarabine; dacarbazine; dactinomycin;daunorubicin hydrochloride; decitabine; dexorrnaplatin; dezaguanine;dezaguanine mesylate; diaziquone; docetaxel; doxorubicin hydrochloride;droloxifene; droloxifene citrate; dromostanolone propionate; duazomycin;edatrexate; eflomithine hydrochloride; elsamitrucin; enloplatin;enpromate; epipropidine; epirubicin hydrochloride; erbulozole;esorubicin hydrochloride; estramustine; estramustine phosphate sodium;etanidazole; etoposide phosphate; etoprine; fadrozole hydrochloride;fazarabine; fenretinide; floxuridine; fludarabine phosphate;fluorouracil; flurocitabine; fosquidone; fostriecin sodium; gemcitabinehydrochloride; hydroxyurea; idarubicin hydrochloride; ifosfamide;ilmofosine; interleukin II (including recombinant interleukin II, orrIL2), interferon alfa-2a; interferon alfa-2b; interferon alfa-nl;interferon alfa-n3; interferon beta-Ia; interferon gamma-Ib; iproplatin;irinotecan hydrochloride; lanreotide acetate; letrozole; leuprolideacetate; liarozole hydrochloride; lometrexol sodium; lomustine;losoxantrone hydrochloride; masoprocol; maytansine; mecchlorethaminehydrochloride; megestrol acetate; melengestrol acetate; melphalan;menogaril; mercaptopurine; methotrexate sodium; metoprine; meturedepa;mitindomide; mitocarcin; mitocromin; mitogillin; mitomalcin; mitomycin;mitusper; mitotane; mitoxantrone hydrochloride; mycophenolic acid;nocodazole; nogalamycin; ormaplatin; oxisuran; pegaspargase; peliomycin;pentamustine; peplomycin sulfate; perfosfarnide; pipobroman; piposulfan;piroxantrone hydrochloride; plicamycin; plomestane; porfimer sodium;porfiromycin; prednimustine; procarbazine hydrochloride; puromycin;puromycin hydrochloride; pyrazofurin; riboprine; rogletimide; safingol;safingol hydrochloride; semustine; simtrazene; sparfosate sodium;sparsornycin; spirogermanium hydrochloride; spiromustine; spiroplatin;streptonigrin; streptozocin; sulofenur; talisomycin; tecogalan sodium;tegafur; teloxantrone hydrochloride; temoporfin; teroxirone;testolactone; thiamiprine; thioguanine; thiotepa; tiazofurin;tirapazamine; toremifene citrate; trestolone acetate; triciribinephosphate; trimetrexate; trimetrexate glucuronate; triptorelin;tubulozole hydrochloride; uracit mustard; uredepa; vapreotide;verteporfln; vinblastine sulfate; vincristine sulfate; vindesine;vindesine sulfate; vinepidine sulfate; vinglycinate sulfate;vinleurosine sulfate; vinorelbine tartrate; vinrosidine sulfate;vinzolidine sulfate; vorozolc; zeniplatin; zinostatin; zorubicinhydrochloride.

Further anti-cancer drugs that can be used in the present inventioninclude, but are not limited to: 17-AAG; 20-epi-1,25-dihydroxyvitaminD3; 5-ethynyluracil; abiraterone; aclarubicin; acylfulvene; adecypenol;adozelesin; aldesleukin; ALL TK antagonists; altretamine; ambamustine;amidox; amifostine; aminolevulinic acid; amrubicin; amsacrine;anagrelide; anastrozole; andrographolide; angiogenesis inhibitors;antagonist D; antagonist G; antarelix; anti-dorsalizing morphogeneticprotein 1; antiandrogen, prostatic carcinoma; antiestrogen;antineoplaston; antisense oligonucleotides; aphidicolin glycinate;apoptosis gene modulators; apoptosis regulators; apurinic acid; ara CDPDL PTBA; arginine deaminase; asulacrine; atamestane; atrimustine;axinastatin 1; axinastatin 2; axinastatin 3; azasetron; azatoxin;azatyrosine; baccatin III derivatives; balanol; batimastat; BCR-ABLantagonists; benzochlorins; benzoylstaurosporine; beta lactamderivatives; beta alethine; betaclarnycin B; betulinic acid; bFGFinhibitor; bicalutamide; bisantrene; bisaziridinylsperrnine; bisnafide;bistratene A; bizelesin; bortezomib; breflate; bropirimine; budotitane;buthionine sulfoximine; calcipotriol; calphostin C; camptothecinderivatives; canarypox IL-2; carboxamide amino triazole;carboxyarnidotriazole; CaRest M3; CARN 700; cartilage derived inhibitor;carzelesin; casein kinase inhibitors; castanospermine; cecropin B;cetrorelix; chlorins; chloroquinoxaline sulfonamide; cicaprost; cisporphyrin; cladribine; clomifene analogues; clotrimazole; collismycin A;collismycin B; combretastatin A4; combretastatin analogue; conagenin;crambescidin 816; crisnatol; cryptophycin 8; cryptophycin A derivatives;curacin A; cyclopentanthraquinones; cycloplatam; cypemycin; cytarabineocfosfate; cytolytic factor; cytostatin; dacliximab; decitabine;dehydrodidemnin B; deslorelin; dexamethasone; dexifosfamide;dexrazoxane; dexveraparnil; diaziquone; didemnin B; didox;diethylnorspermine; dihydro 5 azacytidine; dihydrotaxol, 9; dioxamycin;diphenyl spiromustine; docetaxel; docosanol; dolasetron; doxifluridine;droloxifene; dronabinol; duocarmycin SA; ebselen; ecomustine;edelfosine; edrecolomab; eflomithine; elemene; emitefur; epirubicin;epristeride; estramustine analogue; estrogen agonists; estrogenantagonists; etanidazole; etoposide phosphate; exemestane; fadrozole;fazarabine; fenretinide; filgrastim; finasteride; flavopiridol;flezelastine; fluasterone; fltidarabine; fluorodaunorunieinhydrochloride; forfenimex; formestane; fostriecin; fotemustine;gadolinium texaphyrin; gallium nitrate; galocitabine; ganirelix;gelatinase inhibitors; glutathione inhibitors; hepsulfam; heregulin;hexamethylene bisacetamide; hypericin; ibandronic acid; idarubicin;idoxifene; idramantone; ilmofosine; ilomastat; imidazoacridones;imiquimod; immunostimulant peptides; insulin like growth factor 1receptor inhibitor; interferon agonists; interferons; interleukins;iobenguane; iododoxorubiein; ipomeanol, 4; iroplact; irsogladine;isobengazole; isohomohalicondrin B; itasetron; jasplakinolide;kahalalide F; larnellarin N triacetate; lanreotide; leinamycin;lenograstim; lentinan sulfate; leptolstatin; letrozole; leukemiainhibiting factor; leukocyte alpha interferon;leuprolide+estrogen+progesterone; leuprorelin; levamisole; liarozole;linear polyamine analogue; lipophilic disaccharide peptide; lipophilicplatinum complexes; lissoclinamide 7; lobaplatin; lombricine;lometrexol; lonidamine; losoxantrone; lovastatin; loxoribine;lurtotecan; lutetium texaphyrin; lysofylline; lytic peptides;maitansine; mannostatin A; marimastat; masoprocol; maspin; matrilysininhibitors; matrix metalloproteinase inhibitors; menogaril; merbarone;meterelin; methioninase; metoclopramide; MIF inhibitor; mifepristone;miltefosine; mirimostim; mismatched double stranded RNA; mitoguazone;mitolactol; mitomycin analogues; mitonafide; mitotoxin fibroblast growthfactor saporin; mitoxantrone; mofarotene; molgramostim; monoclonalantibody, human chorionic gonadotrophin; monophosphoryl lipidA+myobacterium cell wall sk; mopidamol; multiple drug resistance geneinhibitor; multiple tumor suppressor 1 based therapy; mustardanti-cancer agent; mycaperoxide B; mycobacterial cell wall extract;myriaporone; N acetyldinaline; N substituted benzamides; nafarelin;nagrestip; naloxone+pentazocine; napavin; naphterpin; nartograstim;nedaplatin; nemorubicin; neridronic acid; neutral endopeptidase;nilutamide; nisamycin; nitric oxide modulators; nitroxide antioxidant;nitrullyn; 06 benzylguanine; octreotide; okicenone; oligonucleotides;onapristone; ondansetron; ondansetron; oracin; oral cytokine inducer;ormaplatin; osaterone; oxaliplatin; oxaunomycin; paclitaxel; paclitaxelanalogues; paclitaxel derivatives; palauamine; palmitoylrhizoxin;pamidronic acid; panaxytriol; panomifene; parabactin; pazelliptine;pegaspargase; peldesine; pentosan polysulfate sodium; pentostatin;pentrozole; perflubron; perfosfamide; perillyl alcohol; phenazinomycin;phenylacetate; phosphatase inhibitors; picibanil; pilocarpinehydrochloride; pirarubicin; piritrexim; placetin A; placetin B;plasminogen activator inhibitor; platinum complex; platinum complexes;platinum triamine complex; porfimer sodium; porfiromycin; prednisone;acridones; prostaglandin J2; proteasome inhibitors; protein A basedimmune modulator; protein kinase C inhibitor; protein kinase Cinhibitors, microalgal; protein tyrosine phosphatase inhibitors; purinenucleoside phosphorylase inhibitors; purpurins; pyrazoloaeridine;pyridoxylated hemoglobin polyoxyethylene conjugate; raf antagonists;raltitrexed; ramosetron; ras farnesyl protein transferase inhibitors;ras inhibitors; ras GAP inhibitor; retelliptine demethylated; rhenium Re186 etidronate; rhizoxin; ribozymes; RH retinamide; rogletimide;rohitukine; romurtide; roquinimex; rubiginone BI; ruboxyl; safingol;saintopin; SarCNU; sarcophytol A; sargramostim; Sdi 1 mimetics;semustine; senescence derived inhibitor 1; sense oligonucleotides;signal transduction inhibitors; signal transduction modulators; singlechain antigen binding protein; sizofiran; sobuzoxane; sodiumborocaptate; sodium phenylacetate; solverol; somatomedin bindingprotein; sonermin; sparfosic acid; spicamycin D; spiromustine;splenopentin; spongistatin 1; squalamine; stem cell inhibitor; stem celldivision inhibitors; stipiamide; stromelysin inhibitors; sulfinosine;superactive vasoactive intestinal peptide antagonist; suradista;suramin; swainsonine; synthetic glycosaminoglycans; tallimustine;tamoxifen methiodide; tauromustine; tazarotene; tecogalan sodium;tegafur; tellurapyrylium; telomerase inhibitors; temoporfin;temozolomide; teniposide; tetrachlorodecaoxide; tetrazomine;thaliblastine; thiocoraline; thrombopoietin; thrombopoietin mimetic;thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroidstimulating hormone; tin ethyl etiopurpurin; tirapazamine; titanocenebichloride; topsentin; toremifene; totipotent stem cell factor;translation inhibitors; tretinoin; triacetyluridine; triciribine;trimetrexate; triptorelin; tropisetron; turosteride; tyrosine kinaseinhibitors; tyrphostins; UBC inhibitors; ubenimex; urogenital sinusderived growth inhibitory factor; urokinase receptor antagonists;vapreotide; variolin B; vector system, erythrocyte gene therapy;velaresol; veramine; verdins; verteporfin; vinorelbine; vinxaltine;vitaxin; vorozole; zanoterone; zeniplatin; zilascorb; zinostatinstimalamer; and PARP inhibitors including, but are not limited toveliparib (ABT-888), oliparib, iniparib, rucaparib, niraparib, BMN 673,E7016 and CEP-9722 and PARP inhibitors described, for example, in U.S.Pat. No. 8,236,802.

It is a further aspect of the invention that the present HDACIs can beadministered in conjunction with chemical agents that are understood tomimic the effects of radiotherapy and/or that function by direct contactwith DNA. Preferred agents for use in combination with the presentHDACIs for treating cancer include, but are not limited tocis-diamminedichloro platinum (II) (cisplatin), doxorubicin,5-fluorouracil, taxol, and topoisomerase inhibitors such as etoposide,teniposide, irinotecan and topotecan.

Additionally, the invention provides methods of treatment of cancerusing the present HDACIs as an alternative to chemotherapy alone orradiotherapy alone where the chemotherapy or the radiotherapy has provenor can prove too toxic, e.g., results in unacceptable or unbearable sideeffects, for the subject being treated. The individual being treatedcan, optionally, be treated with another anticancer treatment modalitysuch as chemotherapy, surgery, or immunotherapy, depending on whichtreatment is found to be acceptable or bearable.

The present HDACIs can also be used in an in vitro or ex vivo fashion,such as for the treatment of certain cancers, including, but not limitedto leukemias and lymphomas, such treatment involving autologous stemcell transplants. This can involve a multi-step process in which thesubject's autologous hematopoietic stem cells are harvested and purgedof all cancer cells, the subject is then administered an amount of apresent HDACI effective to eradicate the subject's remaining bone-marrowcell population, then the stem cell graft is infused back into thesubject. Supportive care then is provided while bone marrow function isrestored and the subject recovers.

The present methods for treating cancer can further comprise theadministration of a present HDACI and an additional therapeutic agent orpharmaceutically acceptable salts or hydrates thereof. In oneembodiment, a composition comprising a present HDACI is administeredconcurrently with the administration of one or more additionaltherapeutic agent(s), which may be part of the same composition or in adifferent composition from that comprising the present HDACI. In anotherembodiment, a present HDACI is administered prior to or subsequent toadministration of another therapeutic agent(s).

In the present methods for treating cancer the other therapeutic agentmay be an antiemetic agent. Suitable antiemetic agents include, but arenot limited to, metoclopromide, domperidone, prochlorperazine,promethazine, chlorpromazine, trimethobenzamide, ondansetron,granisetron, hydroxyzine, acethylleucine monoethanolamine, alizapride,azasetron, benzquinamide, bietanautine, bromopride, buclizine,clebopride, cyclizine, dimenhydrinate, diphenidol, dolasetron,meclizine, methallatal, metopimazine, nabilone, oxyperndyl, pipamazine,scopolamine, sulpiride, tetrahydrocannabinols, thiethylperazine,thioproperazine, and tropisetron.

In a preferred embodiment, the antiemetic agent is granisetron orondansetron. In another embodiment, the other therapeutic agent may bean hematopoietic colony stimulating factor. Suitable hematopoieticcolony stimulating factors include, but are not limited to, filgrastim,sargramostim, molgramostim, and epoietin alfa.

In still another embodiment, the other therapeutic agent may be anopioid or non-opioid analgesic agent. Suitable opioid analgesic agentsinclude, but are not limited to, morphine, heroin, hydromorphone,hydrocodone, oxymorphone, oxycodone, metopon, apomorphine, normorphine,etorphine, buprenorphine, meperidine, lopermide, anileridine,ethoheptazine, piminidine, betaprodine, diphenoxylate, fentanil,sufentanil, alfentanil, remifentanil, levorphanol, dextromethorphan,phenazocine, pentazocine, cyclazocine, methadone, isomethadone, andpropoxyphene. Suitable non-opioid analgesic agents include, but are notlimited to, aspirin, celecoxib, rofecoxib, diclofenac, diflusinal,etodolac, fenoprofen, flurbiprofen, ibuprofen, ketoprofen, indomethacin,ketorolac, meclofenamate, mefanamic acid, nabumetone, naproxen,piroxicam, and sulindac.

In still another embodiment, the other therapeutic agent may be ananxiolytic agent. Suitable anxiolytic agents include, but are notlimited to, buspirene, and benzodiazepines such as diazepam, lorazepam,oxazapam, chlorazepate, clonazepam, chlordiazepoxide and alprazolam.

In addition to treating cancers and sensitizing a cancer cell to thecytotoxic effects of radiotherapy and chemotherapy, the present HDACIsare used in methods of treating diseases, conditions, and injuries tothe central nervous system, such as neurological diseases,neurodegenerative disorders, and traumatic brain injuries (TBIs). Inpreferred embodiments, a present HDACI is capable of crossing the bloodbrain barrier to inhibit HDAC in the brain of the individual.

It has been shown that HDAC6 inhibition protects against neuronaldegeneration and stimulates neurite outgrowth in dorsal root ganglionneurons, therefore indicating methods of treating CNS diseases.Accordingly, present HDACI compounds were examined in a model ofoxidative stress induced by homocysteic acid (HCA). This model leads todepletion of glutathione, the major intracellular antioxidant. HDAC6inhibition rescues neuronal death in this model, possibly by causinghyperacetylation of peroxiredoxins. Previous work reported thatnonselective, hydroxamic acid HDACIs displayed considerable toxicity tothe primary cortical neurons. (A. P. Kozikowski et al., J. Med. Chem.2007, 50, 3054-61.)

The present HDACI compounds also provide a therapeutic benefit in modelsof peripheral neuropathies, such as CMT. HDAC6 inhibitors have beenfound to cross the blood nerve barrier and rescue the phenotype observedin transgenic mice exhibiting symptoms of distal hereditary motorneuropathy. Administration of HDAC6 inhibitors to symptomatic miceincreased acetylated α-tubulin levels, restored proper mitochondrialmotility and axonal transport, and increased muscle re-innervation.Other peripheral neuropathies include, but are not limited to, giantaxonal neuropathy and various forms of mononeuropathies,polyneuropathies, autonomic neuropathies, and neuritis.

The present HDACI compounds also ameliorate associative memory lossfollowing Aβ elevation. In this test, mice were infused with Aβ42 viacannulas implanted into dorsal hippocampus 15 minutes prior to training.The test compounds are dosed ip (25 mg/kg) 2 hours before training. Fearlearning was assessed 24 hours later.

Contextual fear conditioning performed 24 hours after training shows areduction of freezing in Aβ-infused mice compared to vehicle-infusedmice. Treatment with a present compound ameliorates deficit in freezingresponses in Aβ-infused mice, and has no effect in vehicle-infused mice.A test compound alone does not affect the memory performance of themice. In addition, treatment had no effects on motor, sensorial, ormotivational skills assessed using the visible platform test in whichthe compounds are injected twice a day for two days. During theseexperiments, no signs of overt toxicity, including changes in food andliquid intake, weight loss, or changes in locomotion and exploratorybehavior, are observed.

These results demonstrate that the HDACIs of the present invention arebeneficial against impairment of associative memory following Aβelevation.

The present HDACIs therefore are useful for treating a neurologicaldisease by administration of amounts of a present HDACI effective totreat the neurological disease or by administration of a pharmaceuticalcomposition comprising amounts of a present HDACI effective to treat theneurological disease. The neurological diseases that can be treatedinclude, but are not limited to, Huntington's disease, lupus,schizophrenia, multiple sclerosis, muscular dystrophy,dentatorubralpallidoluysian atrophy (DRRLA), spinal and bulbar muscularatrophy (SBMA), and fine spinocerebellar ataxias (SCA1, SCA2, SCA3/MJD(Machado-Joseph Disease), SCA6, and SCAT), drug-induced movementdisorders, Creutzfeldt-Jakob disease, amyotrophic lateral sclerosis,Pick's disease, Alzheimer's disease, Lewy body dementia, cortico basaldegeneration, dystonia, myoclonus, Tourette's syndrome, tremor, chorea,restless leg syndrome, Parkinson's disease, Parkinsonian syndromes,anxiety, depression, psychosis, manic depression, Friedreich's ataxia,Fragile X syndrome, spinal muscular dystrophy, Rett syndrome,Rubinstein-Taybi syndrome, Wilson's disease, multi-infarct state, CMT,GAN and other peripheral neuropathies.

In a preferred embodiment, the neurological disease treated isHuntington's disease, Parkinson's disease, Alzheimer's disease, spinalmuscular atrophy, lupus, or schizophrenia.

A present HDACI also can be used with a second therapeutic agent inmethods of treating conditions, diseases, and injuries to the CNS. Suchsecond therapeutic agents are those drugs known in the art to treat aparticular condition, diseases, or injury, for example, but not limitedto, lithium in the treatment of mood disorders, estradiol benzoate, andnicotinamide in the treatment of Huntington's disease.

The present HDACIs also are useful in the treatment of TBIs. Traumaticbrain injury (TBI) is a serious and complex injury that occurs inapproximately 1.4 million people each year in the United States. TBI isassociated with a broad spectrum of symptoms and disabilities, includinga risk factor for developing neurodegenerative disorders, such asAlzheimer's disease.

TBI produces a number of pathologies including axonal injury, celldeath, contusions, and inflammation. The inflammatory cascade ischaracterized by proinflammatory cytokines and activation of microgliawhich can exacerbate other pathologies. Although the role ofinflammation in TBI is well established, no efficaciousanti-inflammatory therapies are currently available for the treatment ofTBI.

Several known HDAC inhibitors have been found to be protective indifferent cellular and animal models of acute and chronicneurodegenerative injury and disease, for example, Alzheimer's disease,ischemic stroke, multiple sclerosis (MS), Huntington's disease (HD),amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), andspinal and bulbar muscular atrophy (SBMA). A recent study inexperimental pediatric TBI reported a decrease in hippocampal CA3histone H3 acetylation lasting hours to days after injury. These changeswere attributed to documented upstream excitotoxic and stress cascadesassociated with TBI. HDACIs also have been reported to haveanti-inflammatory actions acting through acetylation of non-histoneproteins. The HDAC6 selective inhibitor,4-dimethylamino-N-[5-(2-mercaptoacetylamino)pentyl]benzamide (DMA-PB),was found to be able to increase histone H3 acetylation and reducemicroglia inflammatory response following traumatic brain injury inrats, which demonstrates the utility of HDACIs as therapeutics forinhibiting neuroinflammation associated with TBI.

The present HDACIs therefore also are useful in the treatment ofinflammation and strokes, and in the treatment of autism and autismspectrum disorders. The present HDACIs further can be used to treatparasitic infections, (e.g., malaria, toxoplasmosis, trypanosomiasis,helminthiasis, protozoal infections (see Andrews et al. Int. J.Parasitol. 2000, 30(6), 761-768).

In certain embodiments, the compound of the invention can be used totreat malaria. A present HDACI can be co-administered with anantimalarial compound selected from the group consisting of aryl aminoalcohols, cinchona alkaloids, 4-aminoquinolines, type 1 or type 2 folatesynthesis inhibitors, 8-aminoquinolines, antimicrobials, peroxides,naphthoquinones, and iron chelating agents. The antimalarial compoundcan be, but is not limited to, quinine, quinidine, mefloquine,halfantrine, chloroquine, amodiaquine, proguanil, chloroproquanil,pyrimethamine, primaquine,8-[(4-amino-1-methylbutyl)amino]-2,6-dimethoxy-4-methyl-5-[(3-trifluoromethyl)phenoxy]quinolinesuccinate (WR238,605), tetracycline, doxycycline, clindamycin,azithromycin, fluoroquinolones, artemether, areether, artesunate,artelinic acid, atovaquone, and deferrioxamine. In a preferredembodiment, the antimalarial compound is chloroquine.

The present HDACIs also can be used as imaging agents. In particular, byproviding a radiolabeled, isotopically labeled, or fluorescently-labeledHDACI, the labeled compound can image HDACs, tissues expressing HDACs,and tumors. Labeled HDACIs of the present invention also can imagepatients suffering from a cancer, or other HDAC-mediated diseases, e.g.,stroke, by administration of an effective amount of the labeled compoundor a composition containing the labeled compound. In preferredembodiments, the labeled HDACI is capable of emitting positron radiationand is suitable for use in positron emission tomography (PET).Typically, a labeled HDACI of the present invention is used to identifyareas of tissues or targets that express high concentrations of HDACs.The extent of accumulation of labeled HDACI can be quantified usingknown methods for quantifying radioactive emissions. In addition, thelabeled HDACI can contain a fluorophore or similar reporter capable oftracking the movement of particular HDAC isoforms or organelles invitro.

The present HDACIs useful in the imaging methods contain one or moreradioisotopes capable of emitting one or more forms of radiationsuitable for detection by any standard radiology equipment, such as PET,SPECT, gamma cameras, MRI, and similar apparatus. Preferred isotopesincluding tritium (³H) and carbon (¹¹C). Substituted HDACIs of thepresent invention also can contain isotopes of fluorine (¹⁸F) and iodine(¹²³I) for imaging methods. Typically, a labeled HDACI of the presentinvention contains an alkyl group having a ¹¹C label, i.e., a ¹¹C-methylgroup, or an alkyl group substituted with ¹⁸F, ¹²³I, ¹²⁵I, ¹³¹I, or acombination thereof.

Fluorescently-labeled HDACIs of the present invention also can be usedin the imaging method of the present invention. Such compounds have anFITC,carbocyamine moiety or other fluorophore which will allowvisualization of the HDAC proteins in vitro.

The labeled HDACIs and methods of use can be in vivo, and particularlyon humans, and for in vitro applications, such as diagnostic andresearch applications, using body fluids and cell samples. Imagingmethods using a labeled HDACI of the present invention are discussed inWO 03/060523, designating the U.S. and incorporated in its entiretyherein. Typically, the method comprises contacting cells or tissues witha radiolabeled, isotopically labeled, fluorescently labeled, or tagged(such as biotin tagged) compound of the invention, and making aradiographic, fluorescent, or similar type of image depending on thevisualization method employed, i.e., in regard to radiographic images, asufficient amount to provide about 1 to about 30 mCi of the radiolabeledcompound.

Preferred imaging methods include the use of labeled HDACIs of thepresent invention which are capable of generating at least a 2:1 targetto background ratio of radiation intensity, or more preferably about a5:1, about 10:1, or about 15:1 ratio of radiation intensity betweentarget and background.

In preferred methods, the labeled HDACIs of the present invention areexcreted from tissues of the body quickly to prevent prolonged exposureto the radiation of the radiolabeled compound administered to theindividual. Typically, labeled HDACIs of the present invention areeliminated from the body in less than about 24 hours. More preferably,labeled HDACIs are eliminated from the body in less than about 16 hours,12 hours, 8 hours, 6 hours, 4 hours, 2 hours, 90 minutes, or 60 minutes.Typically, preferred labeled HDACIs are eliminated in about 60 to about120 minutes.

In addition to isotopically labeled and fluorescently labeledderivatives, the present invention also embodies the use of derivativescontaining tags (such as biotin) for the identification of biomoleculesassociated with the HDAC isoforms of interest for diagnostic,therapeutic or research purposes.

The present HDACIs also are useful in the treatment of autoimmunediseases and inflammations. Compounds of the present invention areparticularly useful in overcoming graft and transplant rejections and intreating forms of arthritis.

Despite successes of modern transplant programs, the nephrotoxicity,cardiovascular disease, diabetes, and hyperlipidemia associated withcurrent therapeutic regimens, plus the incidence of post-transplantmalignancies and graft loss from chronic rejection, drive efforts toachieve long-term allograft function in association with minimalimmunosuppression. Likewise, the incidence of inflammatory bowel disease(IBD), including Crohn's disease and ulcerative colitis, is increasing.Animal studies have shown that T regulatory cells (Tregs) expressing theforkhead transcription family member, Foxp3, are key to limitingautoreactive and alloreactive immunity. Moreover, after their inductionby costimulation blockade, immunosuppression, or other strategies, Tregsmay be adoptively transferred to naïve hosts to achieve beneficialtherapeutic effects. However, attempts to develop sufficient Tregs thatmaintain their suppressive functions post-transfer in clinical trialshave failed. Murine studies show that HDACIs limit immune responses, atleast in significant part, by increasing Treg suppressive functions, (R.Tao et al., Nat Med, 13, 1299-1307, (2007)), and that selectivetargeting of HDAC6 is especially efficacious in this regard.

With organ transplantation, rejection begins to develop in the daysimmediately post-transplant, such that prevention rather than treatmentof rejection is a paramount consideration. The reverse applies inautoimmunity, wherein a patient presents with the disease alreadycausing problems. Accordingly, HDAC6−/− mice treated for 14 days withlow-dose RPM (rapamycin) are assessed for displaying signs of toleranceinduction and resistance to the development of chronic rejection, acontinuing major loss of graft function long-term in the clinicaltransplant population. Tolerance is assessed by testing whether micewith long-surviving allografts reject a subsequent third-party cardiacgraft and accept additional donor allografts without anyimmunosuppression, as can occur using a non-selective HDACI plus RPM.These in vivo studies are accompanied by assessment of ELISPOT and MLRactivities using recipient lymphocytes challenged with donor cells.Protection against chronic rejection is assessed by analysis of hostanti-donor humoral responses and analysis of graft transplantarteriosclerosis and interstitial fibrosis in long-surviving allograftrecipients.

The importance of HDAC6 targeting is assessed in additional transplantmodels seeking readouts of biochemical significance, as is monitoredclinically. Thus, the effects of HDAC6 in targeting in renal transplantrecipients (monitoring BUN, proteinuria) and islet allografts(monitoring blood glucose levels) are assessed. Renal transplants arethe most common organ transplants performed, and the kidney performsmultiple functions, e.g., regulating acid/base metabolism, bloodpressure, red cell production, such that efficacy in this modelindicates the utility of HDAC6 targeting. Likewise, islettransplantation is a major unmet need given that clinical isletallografts are typically lost after the first one or two yearspost-transplant. Having a safe and non-toxic means to extend isletsurvival without maintenance CNI therapy would be an important advance.Transplant studies also are strengthened by use of mice with foxedHDAC6. Using existing Foxp3-Cre mice, for example, the effects ofdeletion of HDAC6 just in Tregs is tested. This approach can be extendedto targeting of HDAC6 in T cells (CD4-Cre) and dendritic cells(CD11c-Cre), for example. Using tamoxifen-regulated Cre, the importanceof HDAC6 in induction vs. maintenance of transplants (with implicationsfor short-term vs. maintenance HDAC6I therapy) is assessed byadministering tamoxifen and inducing HDAC6 deletion at varying periodspost-transplant.

Studies of autoimmunity also are undertaken. In this case, interruptionof existing disease is especially important and HDAC6 targeting can beefficacious without any requirement for additional therapy (in contrastto a need for brief low-dose RPM in the very aggressive, fullyMHC-mismatched transplant models). Studies in mice with colitisindicated that HDAC6−/− Tregs were more effective than WT Tregs inregulating disease, and tubacin was able to rescue mice if treatment wasbegun once colitis had developed. These studies are extended byassessing whether deletion of HDAC6 in Tregs (Foxp3/Cre) vs. T cells(CD4=Cre) vs. DC (CD11c-Cre) differentially affect the development andseverity of colitis. Similarly, control of colitis is assessed byinducing HDAC6 deletion at varying intervals after the onset of colitiswith tamoxifen-regulated Cre.

The present compounds are envisioned to demonstrate anti-arthriticefficacy in a collagen-induced arthritis model in DBA1/J mice. In thistest, DBA1/J mice (male, 7-8 weeks) are used, with 8 animals per group.Systemic arthritis is induced with bovine collagen type II and CFA, plusan IFA booster injection on day 21. A present HDACI is dosed at 50 mg/kgand 100 mg/kg on day 28 for 2 consecutive weeks, and the effectsdetermined from the Average Arthritic Score vs. Days of Treatment data.

Despite efforts to avoid graft rejection through host-donor tissue typematching, in the majority of transplantation procedures,immunosuppressive therapy is critical to the viability of the donororgan in the host. A variety of immunosuppressive agents have beenemployed in transplantation procedures, including azathioprine,methotrexate, cyclophosphamide, FK-506, rapamycin, and corticosteroids.

The present HDACIs are potent immunosuppressive agents that suppresshumoral immunity and cell-mediated immune reactions, such as allograftrejection, delayed hypersensitivity, experimental allergicencephalomyelitis, Freund's adjuvant arthritis and graft versus hostdisease. HDACIs of the present invention are useful for the prophylaxisof organ rejection subsequent to organ transplantation, for treatment ofrheumatoid arthritis, for the treatment of psoriasis, and for thetreatment of other autoimmune diseases, such as type I diabetes, Crohn'sdisease, and lupus.

A therapeutically effective amount of a present HDACI can be used forimmunosuppression including, for example, to prevent organ rejection orgraft vs. host disease, and to treat diseases and conditions, inparticular, autoimmune and inflammatory diseases and conditions.Examples of autoimmune and inflammatory diseases include, but are notlimited to, Hashimoto's thyroiditis, pernicious anemia, Addison'sdisease, psoriasis, diabetes, rheumatoid arthritis, systemic lupuserythematosus, dermatomyositis, Sjogren's syndrome, dermatomyositis,lupus erythematosus, multiple sclerosis, myasthenia gravis, Reiter'ssyndrome, arthritis (rheumatoid arthritis, arthritis chronicprogrediente, and arthritis deformans) and rheumatic diseases,autoimmune hematological disorder (hemolytic anaemia, aplastic anaemia,pure red cell anaemia and idiopathic thrombocytopaenia), systemic lupuserythematosus, polychondritis, sclerodoma, Wegener granulamatosis,dermatomyositis, chronic active hepatitis, psoriasis, Steven-Johnsonsyndrome, idiopathic sprue, autoimmune inflammatory bowel disease(ulcerative colitis and Crohn's disease) endocrine opthalmopathy, Gravesdisease, sarcoidosis, primary biliary cirrhosis, juvenile diabetes(diabetes mellitus type I), uveitis (anterior and posterior),keratoconjunctivitis sicca and vernal keratoconjunctivitis, interstitiallung fibrosis, psoriatic arthritis, and glomerulonephritis.

A present HDACI can be used alone, or in conjunction with a secondtherapeutic agent known to be useful in the treatment of autoimmunediseases, inflammations, transplants, and grafts, such as cyclosporin,rapamycin, methotrexate, cyclophosphamide, azathioprine,corticosteroids, and similar agents known to persons skilled in the art.

Additional diseases and conditions mediated by HDACs, and particularlyHDAC6, include, but are not limited to asthma, cardiac hypertrophy,giant axonal neuropathy, mononeuropathy, mononeuritis, polyneuropathy,autonomic neuropathy, neuritis in general, and neuropathy in general.These disease and conditions also can be treated by a method of thepresent invention.

In the present method, a therapeutically effective amount of one or moreHDACI of the present invention, typically formulated in accordance withpharmaceutical practice, is administered to a human being in needthereof. Whether such a treatment is indicated depends on the individualcase and is subject to medical assessment (diagnosis) that takes intoconsideration signs, symptoms, and/or malfunctions that are present, therisks of developing particular signs, symptoms and/or malfunctions, andother factors.

A present HDACI can be administered by any suitable route, for exampleby oral, buccal, inhalation, topical, sublingual, rectal, vaginal,intracisternal or intrathecal through lumbar puncture, transurethral,nasal, percutaneous, i.e., transdermal, or parenteral (includingintravenous, intramuscular, subcutaneous, intracoronary, intradermal,intramammary, intraperitoneal, intraarticular, intrathecal, retrobulbar,intrapulmonary injection and/or surgical implantation at a particularsite) administration. Parenteral administration can be accomplishedusing a needle and syringe or using a high pressure technique.

Pharmaceutical compositions include those wherein a present HDACI ispresent in a sufficient amount to be administered in an effective amountto achieve its intended purpose. The exact formulation, route ofadministration, and dosage is determined by an individual physician inview of the diagnosed condition or disease. Dosage amount and intervalcan be adjusted individually to provide levels of a present HDACI thatis sufficient to maintain therapeutic effects.

Toxicity and therapeutic efficacy of the present HDACI compounds can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., for determining the LD₅₀ (the dose lethal to50% of the population) and the ED₅₀ (the dose therapeutically effectivein 50% of the population). The dose ratio between toxic and therapeuticeffects is the therapeutic index, which is expressed as the ratiobetween LD₅₀ and ED₅₀. Compounds that exhibit high therapeutic indicesare preferred. The data obtained from such procedures can be used informulating a dosage range for use in humans. The dosage preferably lieswithin a range of circulating compound concentrations that include theED₅₀ with little or no toxicity. The dosage can vary within this rangedepending upon the dosage form employed, and the route of administrationutilized. Determination of a therapeutically effective amount is wellwithin the capability of those skilled in the art, especially in lightof the detailed disclosure provided herein.

A therapeutically effective amount of a present HDACI required for usein therapy varies with the nature of the condition being treated, thelength of time that activity is desired, and the age and the conditionof the patient, and ultimately is determined by the attendant physician.Dosage amounts and intervals can be adjusted individually to provideplasma levels of the HDACI that are sufficient to maintain the desiredtherapeutic effects. The desired dose conveniently can be administeredin a single dose, or as multiple doses administered at appropriateintervals, for example as one, two, three, four or more subdoses perday. Multiple doses often are desired, or required. For example, apresent HDACI can be administered at a frequency of: four dosesdelivered as one dose per day at four-day intervals (q4d×4); four dosesdelivered as one dose per day at three-day intervals (q3d×4); one dosedelivered per day at five-day intervals (qd×5); one dose per week forthree weeks (qwk3); five daily doses, with two days rest, and anotherfive daily doses (5/2/5); or, any dose regimen determined to beappropriate for the circumstance.

The dosage of a composition containing a present HDACI, or a compositioncontaining the same, can be from about 1 ng/kg to about 200 mg/kg, about1 μg/kg to about 100 mg/kg, or about 1 mg/kg to about 50 mg/kg of bodyweight. The dosage of a composition may be at any dosage including, butnot limited to, about 1 μg/kg, 10 μg/kg, 25 μg/kg, 50 μg/kg, 75 μg/kg,100 μg/kg, 125 μg/kg, 150 μg/kg, 175 μg/kg, 200 μg/kg, 225 μg/kg, 250μg/kg, 275 μg/kg, 300 μg/kg, 325 μg/kg, 350 μg/kg, 375 μg/kg, 400 μg/kg,425 μg/kg, 450 μg/kg, 475 μg/kg, 500 μg/kg, 525 μg/kg, 550 μg/kg, 575μg/kg, 600 μg/kg, 625 μg/kg, 650 μg/kg, 675 μg/kg, 700 μg/kg, 725 μg/kg,750 μg/kg, 775 μg/kg, 800 μg/kg, 825 μg/kg, 850 μg/kg, 875 μg/kg, 900μg/kg, 925 μg/kg, 950 μg/kg, 975 μg/kg, 1 mg/kg, 5 mg/kg, 10 mg/kg, 15mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50mg/kg, 60 mg/kg, 70 mg/kg, 80 mg/kg, 90 mg/kg, 100 mg/kg, 125 mg/kg, 150mg/kg, 175 mg/kg, or 200 mg/kg. The above dosages are exemplary of theaverage case, but there can be individual instances in which higher orlower dosages are merited, and such are within the scope of thisinvention. In practice, the physician determines the actual dosingregimen that is most suitable for an individual patient, which can varywith the age, weight, and response of the particular patient.

A present HDACI used in a method of the present invention typically isadministered in an amount of about 0.005 to about 500 milligrams perdose, about 0.05 to about 250 milligrams per dose, or about 0.5 to about100 milligrams per dose. For example, a present HDACI can beadministered, per dose, in an amount of about 0.005, 0.05, 0.5, 5, 10,20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500milligrams, including all doses between 0.005 and 500 milligrams.

The HDACIs of the present invention typically are administered inadmixture with a pharmaceutical carrier selected with regard to theintended route of administration and standard pharmaceutical practice.Pharmaceutical compositions for use in accordance with the presentinvention are formulated in a conventional manner using one or morephysiologically acceptable carriers comprising excipients andauxiliaries that facilitate processing of the present HDACIs.

The term “carrier” refers to a diluent, adjuvant, or excipient, withwhich a present HDACI is administered. Such pharmaceutical carriers canbe liquids, such as water and oils, including those of petroleum,animal, vegetable or synthetic origin, such as peanut oil, soybean oil,mineral oil, sesame oil, and the like. The carriers can be saline, gumacacia, gelatin, starch paste, talc, keratin, colloidal silica, urea,and the like. In addition, auxiliary, stabilizing, thickening,lubricating and coloring agents can be used. The pharmaceuticallyacceptable carriers are sterile. Water is a preferred carrier when apresent HDACI is administered intravenously. Saline solutions andaqueous dextrose and glycerol solutions can also be employed as liquidcarriers, particularly for injectable solutions. Suitable pharmaceuticalcarriers also include excipients such as starch, glucose, lactose,sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate,glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol,propylene, glycol, water, ethanol, and the like. The presentcompositions, if desired, can also contain minor amounts of wetting oremulsifying agents, or pH buffering agents.

These pharmaceutical compositions can be manufactured, for example, byconventional mixing, dissolving, granulating, dragee-making,emulsifying, encapsulating, entrapping, or lyophilizing processes.Proper formulation is dependent upon the route of administration chosen.When a therapeutically effective amount of a present HDACI isadministered orally, the composition typically is in the form of atablet, capsule, powder, solution, or elixir. When administered intablet form, the composition additionally can contain a solid carrier,such as a gelatin or an adjuvant. The tablet, capsule, and powdercontain about 0.01% to about 95%, and preferably from about 1% to about50%, of a present HDACI. When administered in liquid form, a liquidcarrier, such as water, petroleum, or oils of animal or plant origin,can be added. The liquid form of the composition can further containphysiological saline solution, dextrose or other saccharide solutions,or glycols. When administered in liquid form, the composition containsabout 0.1% to about 90%, and preferably about 1% to about 50%, byweight, of a present compound.

When a therapeutically effective amount of a present HDACI isadministered by intravenous, cutaneous, or subcutaneous injection, thecomposition is in the form of a pyrogen-free, parenterally acceptableaqueous solution. The preparation of such parenterally acceptablesolutions, having due regard to pH, isotonicity, stability, and thelike, is within the skill in the art. A preferred composition forintravenous, cutaneous, or subcutaneous injection typically contains anisotonic vehicle. A present HDACI can be infused with other fluids overa 10-30 minute span or over several hours.

The present HDACIs can be readily combined with pharmaceuticallyacceptable carriers well-known in the art. Such carriers enable theactive agents to be formulated as tablets, pills, dragees, capsules,liquids, gels, syrups, slurries, suspensions and the like, for oralingestion by a patient to be treated. Pharmaceutical preparations fororal use can be obtained by adding a present HDACI to a solid excipient,optionally grinding the resulting mixture, and processing the mixture ofgranules, after adding suitable auxiliaries, if desired, to obtaintablets or dragee cores. Suitable excipients include, for example,fillers and cellulose preparations. If desired, disintegrating agentscan be added.

A present HDACI can be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection can be presented in unit dosage form, e.g., in ampules orin multidose containers, with an added preservative. The compositionscan take such forms as suspensions, solutions, or emulsions in oily oraqueous vehicles, and can contain formulatory agents such as suspending,stabilizing, and/or dispersing agents.

Pharmaceutical compositions for parenteral administration includeaqueous solutions of the active agent in water-soluble form.Additionally, suspensions of a present HDACI can be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils or synthetic fatty acid esters. Aqueousinjection suspensions can contain substances which increase theviscosity of the suspension. Optionally, the suspension also can containsuitable stabilizers or agents that increase the solubility of thecompounds and allow for the preparation of highly concentratedsolutions. Alternatively, a present composition can be in powder formfor constitution with a suitable vehicle, e.g., sterile pyrogen-freewater, before use.

A present HDACI also can be formulated in rectal compositions, such assuppositories or retention enemas, e.g., containing conventionalsuppository bases. In addition to the formulations described previously,a present HDACI also can be formulated as a depot preparation. Suchlong-acting formulations can be administered by implantation (forexample, subcutaneously or intramuscularly) or by intramuscularinjection. Thus, for example, a present HDACI can be formulated withsuitable polymeric or hydrophobic materials (for example, as an emulsionin an acceptable oil) or ion exchange resins.

In particular, a present HDACI can be administered orally, buccally, orsublingually in the form of tablets containing excipients, such asstarch or lactose, or in capsules or ovules, either alone or inadmixture with excipients, or in the form of elixirs or suspensionscontaining flavoring or coloring agents. Such liquid preparations can beprepared with pharmaceutically acceptable additives, such as suspendingagents. The present HDACIs also can be injected parenterally, forexample, intravenously, intramuscularly, subcutaneously, orintracoronarily. For parenteral administration, the present HDACIs arebest used in the form of a sterile aqueous solution which can containother substances, for example, salts or monosaccharides, such asmannitol or glucose, to make the solution isotonic with blood.

As an additional embodiment, the present invention includes kits whichcomprise one or more compounds or compositions packaged in a manner thatfacilitates their use to practice methods of the invention. In onesimple embodiment, the kit includes a compound or composition describedherein as useful for practice of a method (e.g., a compositioncomprising a present HDACI and an optional second therapeutic agent),packaged in a container, such as a sealed bottle or vessel, with a labelaffixed to the container or included in the kit that describes use ofthe compound or composition to practice the method of the invention.Preferably, the compound or composition is packaged in a unit dosageform. The kit further can include a device suitable for administeringthe composition according to the intended route of administration, forexample, a syringe, drip bag, or patch. In another embodiment, thepresent compound is a lyophilate. In this instance, the kit can furthercomprise an additional container which contains a solution useful forthe reconstruction of the lyophilate.

Prior HDACIs possessed properties that hindered their development astherapeutic agents. In accordance with an important feature of thepresent invention, the present HDACIs were synthesized and evaluated asinhibitors for HDAC. The present compounds demonstrate an increasedHDAC6 potency and selectivity against class 1 HDAC and with improvementsin BEI relative to prior compounds. The improved properties of thepresent compounds, particularly the increase in BEI and reduced potencyat HDAC8, indicate that the present compounds are useful forapplications such as, but not limited to, immunosuppresssive andneuroprotective agents.

What is claimed:
 1. A method of inhibiting HDAC, comprisingadministering to an individual in need thereof a therapeuticallyeffective amount of a compound of formula: (Ia):

wherein A is selected from the group consisting of —C(═O)NHheteroaryl,—NHC(═O)heteroaryl, aryl, heteroaryl, and

 wherein B and C are, independently, aryl or heterocyclyl and n is 2 or3; or a pharmaceutically acceptable salt thereof; or (Ib):

wherein A is selected from the group consisting of, —NHC(═O)alkyl,—NHC(═O)cycloalkyl, —NHC(═O)heteroaryl, aryl, and heteroaryl; n is 0 or1; and p is 0 or 1; or a pharmaceutically acceptable salt thereof.
 2. Amethod of inhibiting HDAC comprising administering to an individual inneed thereof a therapeutically effective amount of a compound selectedfrom the group consisting of

or a pharmaceutically acceptable salt thereof; wherein Boc istert-butoxycarbonyl; and Bz is benzoyl.
 3. The method of claim 1,wherein the HDAC is HDAC6.
 4. The method of claim 1, further comprisingadministering a therapeutically effective amount of a second therapeuticagent.
 5. The method of claim 1, wherein the individual in need thereofis a cancer patient.
 6. The method of claim 4 wherein the secondtherapeutic agent is selected from anticancer agents, antiemetic agents,hematopoietic colony stimulating factor, opioid analgesics agents, andanxiolytic agents.
 7. The method of claim 1, wherein the individual inneed thereof suffers from a disease or condition selected from aneurological disease, a neurodegenerative disorder, peripheralneuropathy, a traumatic brain injury, stroke, a parasitic infection,autism, or an autism spectrum disorder.
 8. The method of claim 7,wherein the disease or condition is a neurological disease, aneurodegenerative disorder, peripheral neuropathy, or a traumatic braininjury selected from Huntington's disease, Parkinson's disease,Alzheimer's disease, ischemic stroke, multiple sclerosis, amyotrophiclateral sclerosis (ALS), spinal bulbar muscular atrophy (SBMA), spinalmuscular atrophy, lupus, or schizophrenia.
 9. The method of claim 7,wherein the disease or condition is a parasitic infection selected fromthe group consisting of malaria, toxoplasmosis, trypanosomiasis,helminthiasis, and a protozoal infection.
 10. The method of claim 9,further comprising coadministering to the individual an antimalarialcompound selected from the group consisting of an aryl amino alcohol, acinchona alkaloid, a 4-aminoquinoline, a type 1 or type 2 folatesynthesis inhibitor, an 8-aminoquinoline, an antimicrobial, a peroxide,a naphthoquinone, and an iron-chelating agent.
 11. The method of claim10, wherein the antimalarial compound is selected from the groupconsisting of quinine, quinidine, mefloquine, halofantrine, chloroquine,amodiaquine, proguanil, chloroproguanil, pyrimethamine, primaquine,8-[(4-amino-l-methylbutyl)aminb]-2,6-dimethoxy-4-methyl-5-[(3-trifluoromethyl)phenoxy]quinolinesuccinate (WR238,605), tetracycline, doxycycline, clindamycin,azithromycin, fluoroquinolones, artemether, arteether, artesunate,artelinic acid, atovaquone, and desferrioxamine.
 12. The method of claim11, wherein the antimalarial compound is chloroquine.
 13. A method ofincreasing sensitivity of a cancer cell to cytotoxic effects of aradiotherapy and/or a chemotherapy comprising contacting the cell with acompound of formula: (Ia):

wherein A is selected from the group consisting of —C(═O)NHheteroaryl,—NHC(═O)heteroaryl, aryl, heteroaryl, and

 wherein B and C are, independently, aryl or heterocyclyl and n is 0-4;or a pharmaceutically acceptable salt thereof; or (Ib):

wherein A is selected from the group consisting of, —NHC(═O)alkyl,—NHC(═O)cycloalkyl, —NHC(═O)heteroaryl, aryl, and heteroaryl; n is 0 or1; and p is 0 or 1; or a pharmaceutically acceptable salt thereof, in anamount sufficient to increase the sensitivity of the cell to theradiotherapy and/or the chemotherapy.
 14. The method of claim 13,wherein the cell is an in vivo cell.
 15. The method of claim 1, whereinindividual in need thereof is suffering from a disease or conditionsselected from Hashimoto's thyroiditis, pernicious anemia, Addison'sdisease, psoriasis, diabetes, rheumatoid arthritis, systemic lupuserythematosus, dermatomyositis, Sjogren's syndrome, dermatomyositis,lupus erythematosus, multiple sclerosis, myasthenia gravis, Reiter'ssyndrome, arthritis, rheumatic disease, hemolytic anemia, aplasticanemia, pure red cell anemia, idiopathic thrombocytopaenia, systemiclupus erythematosus, polychondritis, sclerodoma, Wegener granulomatosis,dermatomyositis, chronic active hepatitis, Steven-Johnson syndrome,idiopathic sprue, inflammatory bowel disease, endocrine ophthalmopathy,Graves disease, sarcoidosis, primary biliary cirrhosis, juvenilediabetes, uveitis, keratoconjunctivitis sicca, and glomerulonephritis.16. A method of treating pancreatic cancer, comprising administering toan individual in need thereof a therapeutically effective amount of acompound of formula: (Ia):

wherein A is selected from the group consisting of —C(═O)NHheteroaryl,—NHC(═O)heteroaryl, aryl, heteroaryl, and

 wherein B and C are, independently, aryl or heterocyclyl and n is 2 or3; or a pharmaceutically acceptable; or (Ib):

wherein A is selected from the group consisting of, —NHC(═O)alkyl,—NHC(═O)cycloalkyl, —NHC(═O)heteroaryl, aryl, and heteroaryl; n is 0 or1; and p is 0 or 1; or a pharmaceutically acceptable salt thereof.